Bi-specific aptamer

ABSTRACT

Provided are aptamers and aptamer compositions and particularly, although not exclusively, to a bi-specific aptamer capable of binding a tumor cell antigen and an immune cell surface protein.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/297,487, filed Feb. 19, 2016, the content of which is incorporatedherein by reference in its entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

NOT APPLICABLE.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

The Sequence Listing written in file 48440-608001WO_ST25.TXT, created onFeb. 13, 2017, 36,031 bytes, machine format IBM-PC, MS Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to aptamers and aptamer compositions andparticularly, although not exclusively, to a bi-specific aptamer capableof binding a tumor cell antigen and an immune cell surface protein.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common causeof cancer death in the United States, accounting for 30,000 deathsyearly in the US (Jemal, A. et al. Cancer statistics, 2009. CA Cancer JClin 59, 225-249 (2009)). Despite great efforts to improve treatment forpatients with pancreatic cancer, limited progress has been made(Stathis, A. & Moore, M. J. Advanced pancreatic carcinoma: currenttreatment and future challenges. Nature reviews. Clinical oncology 7,163-172 (2010); Pancreatic cancer in the UK. Lancet 378, 1050 (2011)).Although much research has been conducted to develop improved systemictherapies for pancreatic cancer, gemcitabine as a single agent givenpostoperatively remains the current standard of care. Combinations withother chemotherapeutic drugs or biological agents given as a palliativesetting for unresectable pancreatic cancer or adjuvant setting followingresection have resulted in limited improvement (Klinkenbijl, J. H. etal. Adjuvant radiotherapy and 5-fluorouracil after curative resection ofcancer of the pancreas and periampullary region: phase III trial of theEORTC gastrointestinal tract cancer cooperative group. Annals of surgery230, 776-782; discussion 782-774 (1999); Neoptolemos, J. P. et al. Arandomized trial of chemoradiotherapy and chemotherapy after resectionof pancreatic cancer. The New England Journal of Medicine 350, 1200-1210(2004); Oettle, H. et al. Adjuvant chemotherapy with gemcitabine vsobservation in patients undergoing curative-intent resection ofpancreatic cancer: a randomized controlled trial. JA1UA: the journal ofthe American 1 Uedical Association 297, 267-277 (2007)). The 5 yearsurvival of patients with pancreatic cancer, despite numerous phase 3trials, remains less than 5% after resection (Vincent, A., Herman, J.,Schulick, R., Hruban, R. H. & Goggins, M. Pancreatic cancer. Lancet 378,607-620 (2011); Alexakis, N. et al. Current standards of surgery forpancreatic cancer. The British Journal of Surgery 91, 1410-1427 (2004);Ghaneh, P., Costello, E. & Neoptolemos, J. P. Biology and management ofpancreatic cancer. Gut 56, 1134-1152 (2007)). The majority of patientswill present with either local or systemic recurrence within 2 yearsfollowing resection and postoperative adjuvant chemotherapy.

Currently, the most effective single agent gemcitabine achieves animproved 1-year survival rate from 16 to 19%. The addition of Tarceva®(erlotinib) in a randomized study added a median of 11 days to overallsurvival (Cunningham, D. et al. Phase III randomized comparison ofgemcitabine versus gemcitabine plus capecitabine in patients withadvanced pancreatic cancer. Journal of clinical oncology: officialjournal of the American Society of Clinical Oncology 27, 5513-5518(2009). Heinemann, V., Haas, M. & Boeck, S. Systemic treatment ofadvanced pancreatic cancer. Cancer treatment reviews 38, 843-853(2012)). This limitation of conventional treatment is due to theprofound resistance of PDAC cells towards anti-cancer drugs emergingfrom the efficient protection against chemotherapeutic drugs (Wong, H.H.& Lemoine, N. R. Pancreatic cancer: molecular pathogenesis and newtherapeutic targets. Nat Rev Gastroenterol Hepatol 6, 412-422 (2009);Fulda, S. Apoptosis pathways and their therapeutic exploitation inpancreatic cancer. J Cell 1Uol 1Ued 13, 1221-1227 (2009)). Therefore, itis imperative to develop new therapeutic strategies for this devastatingdisease. Provided herein are solutions to these and other problems inthe art.

BRIEF SUMMARY OF THE INVENTION

The inventors have provided a nucleic acid composition capable ofbinding to a tumor cell antigen and an immune cell surface protein. Thisbi-specific aptamer is a capable of binding to cell surface proteinspresent on a tumor cell and simultaneously binding to a cell surfaceprotein on an immune cell, e.g. lymphocyte, T-cell, T-helper cell,cytotoxic T cell, CD8+ T-cell, CD4+ T-cell, B cell, leukocyte,macrophage, neutrophil, dendritic cell, preferably a T-cell. Thebi-specific aptamer forms a bridge between the two cell types and allowsfor an enhanced immune response to the tumor cell, improved T-cellengagement and improved tumor cell killing. The bi-specific aptamer isformed from a nucleic acid compound (aptamer) capable of binding to atumor cell antigen in complex with a nucleic acid compound (aptamer)capable of binding to an immune cell surface protein. The two aptamercomponents may form the bi-specific aptamer complex through covalent ornon-covalent association. Bi-specific aptamers according to the presentinvention may comprise a complex of a tumor cell binding aptamer and animmune cell binding aptamer.

The bi-specific aptamer is useful in therapeutic, diagnostic and imagingapplications. Pharmaceutical, diagnostic and imaging compositionscomprising the bi-specific aptamer are provided. Methods of treatment,particularly of cancer, comprising administering the bi-specific aptamerto a subject in need of treatment are also provided. Diagnostic andimaging methods involving the use of the bi-specific aptamer are alsoprovided. The bi-specific aptamer may also be conjugated to a compoundmoiety, which may be a therapeutic, diagnostic or imaging moiety.

In one aspect of the present invention a bi-specific aptamer capable ofbinding a tumor cell antigen and an immune cell surface protein isprovided.

In some embodiments the tumor cell antigen is HSP70, vimentin, HSP90,TfR or PDGFR-a.

In some embodiments the immune cell surface protein is selected from thegroup consisting of CCR5, CCR7, CD2, CD3, CD4, CD7, CD8, PD-1, CTLA4.

In another aspect of the present invention a bi-specific aptamer capableof binding HSP70 and an immune cell surface protein is provided. Thenucleic acid sequence of three HSP70 binding aptamers is shown in FIG.10 as SEQ ID NOs:1, 2 and 4.

In another aspect of the present invention a bi-specific aptamer capableof binding vimentin and an immune cell surface protein is provided. Thenucleic acid sequence of a vimentin binding aptamer is shown in FIG. 10as SEQ ID NO:3.

In another aspect of the present invention a bi-specific aptamer capableof binding HSP90 and an immune cell surface protein is provided. Thenucleic acid sequence of three HSP90 binding aptamers is shown in FIG.10 as SEQ ID NOs:5, 6 and 7.

In another aspect of the present invention a bi-specific aptamer capableof binding TfR and an immune cell surface protein is provided.

In another aspect of the present invention a bi-specific aptamer capableof binding PDGFR-a and an immune cell surface protein is provided.

In some embodiments the immune cell surface protein is selected from thegroup consisting of CCR5, CCR7, CD2, CD3, CD4, CD7, CD8, PD-1, CTLA4.

In another aspect of the present invention a bi-specific aptamer capableof binding a cancer cell and an immune cell is provided. In someembodiments a bi-specific aptamer capable of binding a pancreatic cancercell and an immune cell is provided. The nucleic acid sequence ofpancreatic cancer binding aptamers is shown in FIG. 10 as SEQ ID NOs:1to 8. The immune cell may be a lymphocyte, white blood cell, T-cell(thymocyte), T-helper cell, cytotoxic T-cell, CD8+ T-cell, CD4+ T-cell,memory T-cell, suppressor T-cell, natural killer T-cell, gamma deltaT-cell, B cell, natural killer cell, leukocyte, macrophage, neutrophil,dendritic cell. In some embodiments the immune cell may be a T-cell,preferably a CD8+ T-cell and/or a CD4+ T-cell. In some embodiments theimmune cell is a cytotoxic T-cell.

In another aspect of the present invention a bi-specific aptamer capableof binding HSP70 and CCR5 is provided. In another aspect of the presentinvention a bi-specific aptamer capable of binding HSP70 and CCR7 isprovided. In another aspect of the present invention a bi-specificaptamer capable of binding HSP70 and CD2 is provided. In another aspectof the present invention a bi-specific aptamer capable of binding HSP70and CD3 is provided. In another aspect of the present invention abi-specific aptamer capable of binding HSP70 and CD4 is provided. Inanother aspect of the present invention a bi-specific aptamer capable ofbinding HSP70 and CD7 is provided. In another aspect of the presentinvention a bi-specific aptamer capable of binding HSP70 and CD8 isprovided. In another aspect of the present invention a bi-specificaptamer capable of binding HSP70 and PD-1 is provided. In another aspectof the present invention a bi-specific aptamer capable of binding HSP70and CTLA4 is provided. In some preferred embodiments the HSP70 ismHSP70.

In another aspect of the present invention a bi-specific aptamer capableof binding vimentin and CCR5 is provided. In another aspect of thepresent invention a bi-specific aptamer capable of binding vimentin andCCR7 is provided. In another aspect of the present invention abi-specific aptamer capable of binding vimentin and CD2 is provided. Inanother aspect of the present invention a bi-specific aptamer capable ofbinding vimentin and CD3 is provided. In another aspect of the presentinvention a bi-specific aptamer capable of binding vimentin and CD4 isprovided. In another aspect of the present invention a bi-specificaptamer capable of binding vimentin and CD7 is provided. In anotheraspect of the present invention a bi-specific aptamer capable of bindingvimentin and CD8 is provided. In another aspect of the present inventiona bi-specific aptamer capable of binding vimentin and PD-1 is provided.In another aspect of the present invention a bi-specific aptamer capableof binding vimentin and CTLA4 is provided.

In another aspect of the present invention a bi-specific aptamer capableof binding HSP90 and CCR5 is provided. In another aspect of the presentinvention a bi-specific aptamer capable of binding HSP90 and CCR7 isprovided. In another aspect of the present invention a bi-specificaptamer capable of binding HSP90 and CD2 is provided. In another aspectof the present invention a bi-specific aptamer capable of binding HSP90and CD3 is provided. In another aspect of the present invention abi-specific aptamer capable of binding HSP90 and CD4 is provided. Inanother aspect of the present invention a bi-specific aptamer capable ofbinding HSP90 and CD7 is provided. In another aspect of the presentinvention a bi-specific aptamer capable of binding HSP90 and CD8 isprovided. In another aspect of the present invention a bi-specificaptamer capable of binding HSP90 and PD-1 is provided. In another aspectof the present invention a bi-specific aptamer capable of binding HSP90and CTLA4 is provided.

In another aspect of the present invention a bi-specific aptamer capableof binding a pancreatic cancer cell and CCR5 is provided. In anotheraspect of the present invention a bi-specific aptamer capable of bindinga pancreatic cancer cell and CCR7 is provided. In another aspect of thepresent invention a bi-specific aptamer capable of binding a pancreaticcancer cell and CD2 is provided. In another aspect of the presentinvention a bi-specific aptamer capable of binding a pancreatic cancercell and CD3 is provided. In another aspect of the present invention abi-specific aptamer capable of binding a pancreatic cancer cell and CD4is provided. In another aspect of the present invention a bi-specificaptamer capable of binding a pancreatic cancer cell and CD7 is provided.In another aspect of the present invention a bi-specific aptamer capableof binding a pancreatic cancer cell and CD8 is provided. In anotheraspect of the present invention a bi-specific aptamer capable of bindinga pancreatic cancer cell and PD-1 is provided. In another aspect of thepresent invention a bi-specific aptamer capable of binding a pancreaticcancer cell and CTLA4 is provided.

In another aspect of the present invention a bi-specific aptamer capableof binding TfR and CCR5 is provided. In another aspect of the presentinvention a bi-specific aptamer capable of binding TfR and CCR7 isprovided. In another aspect of the present invention a bi-specificaptamer capable of binding TfR and CD2 is provided. In another aspect ofthe present invention a bi-specific aptamer capable of binding TfR andCD3 is provided.

In another aspect of the present invention a bi-specific aptamer capableof binding TfR and CD4 is provided. In another aspect of the presentinvention a bi-specific aptamer capable of binding TfR and CD7 isprovided. In another aspect of the present invention a bi-specificaptamer capable of binding TfR and CD8 is provided. In another aspect ofthe present invention a bi-specific aptamer capable of binding TfR andPD-1 is provided. In another aspect of the present invention abi-specific aptamer capable of binding TfR and CTLA4 is provided.

In another aspect of the present invention a bi-specific aptamer capableof binding PDGFR-a and CCR5 is provided. In another aspect of thepresent invention a bi-specific aptamer capable of binding PDGFR-a andCCR7 is provided. In another aspect of the present invention abi-specific aptamer capable of binding PDGFR-a and CD2 is provided. Inanother aspect of the present invention a bi-specific aptamer capable ofbinding PDGFR-a and CD3 is provided. In another aspect of the presentinvention a bi-specific aptamer capable of binding PDGFR-a and CD4 isprovided. In another aspect of the present invention a bi-specificaptamer capable of binding PDGFR-a and CD7 is provided. In anotheraspect of the present invention a bi-specific aptamer capable of bindingPDGFR-a and CD8 is provided. In another aspect of the present inventiona bi-specific aptamer capable of binding PDGFR-a and PD-1 is provided.In another aspect of the present invention a bi-specific aptamer capableof binding PDGFR-a and CTLA4 is provided.

In some aspects of the present invention the bi-specific aptamercomprises the nucleic acid sequence of one of SEQ ID NOs:1 to 8 or anucleic acid sequence having at least 80% sequence identity to one ofSEQ ID NOs:1 to 8. The nucleic acid sequence of the region of theaptamer capable of binding to HSP70, vimentin, HSP90 or a cancer cell,e.g. pancreatic cancer cell, may comprise, or consist of, one of SEQ IDNOs:1 to 8 or a nucleic acid sequence having at least 80% sequenceidentity to one of SEQ ID NOs:1 to 8.

In some aspects of the present invention the bi-specific aptamercomprises the nucleic acid sequence of one of SEQ ID NOs:1, 2 and 4 or anucleic acid sequence having at least 80% sequence identity to one ofSEQ ID NOs:1, 2 and 4. The nucleic acid sequence of the region of theaptamer capable of binding to HSP70 or a cancer cell, e.g. pancreaticcancer cell, may comprise, or consist of, one of SEQ ID NOs:1, 2 and 4or a nucleic acid sequence having at least 80% sequence identity to oneof SEQ ID NOs:1, 2 and 4.

In some embodiments the bi-specific aptamer comprises the nucleic acidsequence of SEQ ID NO:3 or a nucleic acid sequence having at least 80%sequence identity to SEQ ID NO:3. The nucleic acid sequence of theregion of the aptamer capable of binding to vimentin or a cancer cell,e.g. pancreatic cancer cell, may comprise, or consist of, SEQ ID NO:3 ora nucleic acid sequence having at least 80% sequence identity to SEQ IDNO:3.

In some aspects of the present invention the bi-specific aptamercomprises the nucleic acid sequence of one of SEQ ID NOs:5, 6 and 7 or anucleic acid sequence having at least 80% sequence identity to one ofSEQ ID NOs:5, 6 and 7. The nucleic acid sequence of the region of theaptamer capable of binding to HSP90 or a cancer cell, e.g. pancreaticcancer cell, may comprise, or consist of, one of SEQ ID NOs:5, 6 and 7or a nucleic acid sequence having at least 80% sequence identity to oneof SEQ ID NOs:5, 6 and 7.

In some aspects of the present invention the bi-specific aptamercomprises the nucleic acid sequence of one of SEQ ID NOS:28, 29 and 30or a nucleic acid sequence having at least 80% sequence identity to oneof SEQ ID NOs:28, 29 and 30. The nucleic acid sequence of the region ofthe aptamer capable of binding to TfR or a cancer cell, e.g. pancreaticcancer cell, may comprise, or consist of, one of SEQ ID NOs:28, 29 and30 or a nucleic acid sequence having at least 80% sequence identity toone of SEQ ID NOs:28, 29 and 30.

In some aspects of the present invention the bi-specific aptamercomprises the nucleic acid sequence of one of SEQ ID NOs:31 and 32 or anucleic acid sequence having at least 80% sequence identity to one ofSEQ ID NOs:31 and 32. The nucleic acid sequence of the region of theaptamer capable of binding to PDGFR-a or a cancer cell, e.g. pancreaticcancer cell, may comprise, or consist of, one of SEQ ID NOs:31 and 32 ora nucleic acid sequence having at least 80% sequence identity to one ofSEQ ID NOs:31 and 32.

In some embodiments the bi-specific aptamer comprises the nucleic acidsequence of one of SEQ ID NOs:9 to 16 or a nucleic acid sequence havingat least 80% sequence identity to one of SEQ ID NOs:9 to 16. The nucleicacid sequence of the region of the aptamer capable of binding to CCR5may comprise, or consist of, one of SEQ ID NOs:9 to 16 or a nucleic acidsequence having at least 80% sequence identity to one of SEQ ID NOs:9 to16.

The bi-specific aptamer may comprise a complex, preferably anon-covalent complex, of SEQ ID Nos 17 and 18, or a complex of a nucleicacid sequence having at least 80% sequence identity to SEQ ID NO: 17with a nucleic acid sequence having at least 80% sequence identity toSEQ ID NO: 18.

In some aspects of the present invention a bi-specific aptamercomprises, or consists of, a nucleic acid sequence selected from one of:SEQ ID NOs:1, 2 or 4; SEQ ID NO:3; SEQ ID NOs:5, 6 or 7; SEQ ID NOs:28,29 or 30; SEQ ID NOs:31 or 32, or a nucleic acid sequence having atleast 80% sequence identity to one of said sequences, and a nucleic acidsequence comprising, or consisting of, one of SEQ ID NOs:9 to 16, or anucleic acid sequence having at least 80% sequence identity to one ofsaid sequences.

In some aspects of the present invention a bi-specific aptamer comprisesone of SEQ ID NOs:17, 19 to 24, and 33, or a nucleic acid sequencehaving at least 80% sequence identity to said sequence. In some aspectsof the present invention a bi-specific aptamer comprises one of SEQ IDNOs:18, 37 and 38, or a nucleic acid sequence having at least 80%sequence identity to said sequence. In some aspects of the presentinvention a bi-specific aptamer comprises a complex of one of one of SEQID NOs: 17, 19 to 24, and 33 or a nucleic acid sequence having at least80% sequence identity to said sequence and one of SEQ ID NOs:18, 37 and38 or a nucleic acid sequence having at least 80% sequence identity tosaid sequence.

In some embodiments one or more bases or nucleotides are chemicallymodified. In some embodiments one or more nucleotides are chemicallymodified at the 2′ position of ribose. Nucleic acid sequences of theaptamers according to the present invention may be RNA and/or maycomprise 2′-fluoro modified pyrimidine and/or may comprise2′-O-methylated purine.

In another aspect of the present invention a complex, preferably anon-covalent complex, of a bi-specific aptamer and a tumor cellexpressing a tumor cell antigen to which the bi-specific aptamer iscapable of binding is provided. In another aspect of the presentinvention a complex, preferably a non-covalent complex, of a bi-specificaptamer and an immune cell expressing an immune cell surface protein towhich the bi-specific aptamer is capable of binding is provided. Inanother aspect of the present invention a complex, preferably anon-covalent complex, of a bi-specific aptamer, a tumor cell expressinga tumor cell antigen to which the bi-specific aptamer is capable ofbinding and an immune cell expressing an immune cell surface protein towhich the bi-specific aptamer is capable of binding is provided. Theimmune cell may be a T-cell, e.g. CD8+ and/or CD4+ T-cell or cytotoxicT-cell. In some embodiments, the complex is formed in vitro, and isoptionally isolated. In other embodiments the complex may be formed invivo. The complex preferably comprises the bi-specific aptamer bound toone or both of the tumor cell and immune cell. The tumor cell may be ofany type of cancer, as described herein. In some embodiments it is apancreatic cancer cell or glioblastoma cell.

In another aspect of the present invention a pharmaceutical compositioncomprising a bi-specific aptamer according to the present invention anda pharmaceutically acceptable carrier, diluent or excipient is provided.

In another aspect of the present invention a bi-specific aptameraccording to the present invention is provided for use in a method ofmedical treatment.

In another aspect of the present invention a bi-specific aptameraccording to the present invention is provided for use in a method oftreatment of cancer.

In another aspect of the present invention the use of a bi-specificaptamer according to the present invention in the manufacture of amedicament for use in a method of medical treatment is provided.

In another aspect of the present invention the use of a bi-specificaptamer according to the present invention in the manufacture of amedicament for use in a method of treatment of cancer is provided.

In another aspect of the present invention a method of treatment ofcancer in a subject in need of treatment is provided, the methodcomprising administering a therapeutically effective amount of abi-specific aptamer according to the subject.

In some embodiments, the cancer is a pancreatic cancer. In someembodiments the cancer overexpresses at least one of HSP70, vimentin,HSP90, TfR or PDGFR-a.

In another aspect of the present invention a method of selecting asubject for treatment of cancer with a therapeutically effective amountof a bi-specific aptamer according to the present invention is provided,the method comprising determining, in vitro, whether cells of a cancerin the subject overexpress at least one of HSP70, vimentin, HSP90, TfRor PDGFR-a.

In some embodiments the aptamers or bi-specific aptamers according tothe present invention are capable of internalising into a cell followingbinding to a cell surface target molecule. Such aptamers are useful inmethods of delivering a compound moiety to the cell, where the compoundmoiety is conjugated to the aptamer.

Aptamers and bi-specific aptamers according to the present invention mayalso inhibit proliferation of cells in vitro or in vivo. This mayinvolve inhibition of cancer/tumor cell proliferation. This may be acytostatic effect, but may also be a cytotoxic effect. As such, aptamersaccording to the present invention are provided for use in methods ofmedical treatment where inhibition of cell proliferation is useful fortreatment of a disease.

In some embodiments an aptamer, bi-specific aptamer or nucleic acidcompound may further comprises a compound moiety covalently attached tosaid nucleic acid sequence. The compound moiety may be a therapeuticmoiety or an imaging moiety.

The therapeutic moiety may be a nucleic acid moiety, an antibody, apeptide moiety or a small molecule drug moiety. The therapeutic moietyis may be an activating nucleic acid moiety or an antisense nucleic acidmoiety. The therapeutic moiety may be an miRNA moiety, mRNA moiety,siRNA moiety or an saRNA moiety. The therapeutic moiety may be an siRNAmoiety or saRNA moiety. The therapeutic moiety may be an anticanceragent moiety. The therapeutic moiety may be a C/EBPalpha saRNA moiety ora KRAS siRNA moiety. The imaging agent moiety may be a bioluminescentmolecule, a photoactive molecule, a metal or a nanoparticle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Diagram illustrating bi-specific aptamer comprising CCR5aptamer, spacer and tP19 aptamer.

FIG. 2. Diagram illustrating linkage of tP19 and CCR5 aptamer using‘sticky end’ complementary nucleic acid sequences.

FIG. 3. Photograph showing isolation of tP19 aptamer, CCR5 aptamer andtP19-CCR5 bi-specific aptamer by gel electrophoresis.

FIG. 4. Conjugates of bi-specific aptamers revealed by electrophoresis.

FIG. 5. Diagram illustrating use of Cy3 and FAM labelling to identifylocation of bi-specific aptamer during confocal microscopy.

FIG. 6. Micrograph showing lack of association of T-cells (small cells)with PANC-1 cells (large cell) in absence of bi-specific aptamer.

FIG. 7. Sequential images of T cells probing tumor cells. CD8 T cellswere incubated with PANC-1 in the presence of P19-CCR5 bi-specificaptamer. Arrows to smaller cells indicates CD8 T cells. Arrows to largercells indicates PANC-1 cells.

FIG. 8. Micrograph illustrating formation of immunological synapse.

FIG. 9. Chart showing results of bi-specific aptamer cytotoxic T-cellassay.

FIG. 10. Nucleic acid sequences of aptamers truncated P19 (tP19), fulllength P19, P15, P1, P11, P7 and P6 and consensus sequence SEQ ID NO:8.This series of aptamers is described in WO2013/154735. P19 and P1 bindHSP70. P15 binds vimentin. P11, P7 and P6 bind HSP90.

FIG. 11. Nucleic acid sequence of CCR5 aptamers.

FIGS. 12A-12B. Nucleic acid sequence of components of bi-specificaptamer targeting mHSP70 and CCR5. Bi-specific aptamer is a non-covalentcomplex of sequences depicted in (A) and (B). (FIG. 12A) Nucleic acidsequence of truncated P19 aptamer capable of binding to mHSP70conjugated to a sticky sequence (bold) with intermediate seven C3 carbonspacer (each C3 carbon represented by “o”). fC and fU indicates2′-fluoro pyrimidine modification, mA and mG indicates 2′-OMe purinemodification. (FIG. 12B) Nucleic acid sequence of CCR5 aptamerconjugated to a sticky sequence (bold) with intermediate five C3 carbonspacer (each C3 carbon represented by “o”). fC and fU indicates2′-fluoro pyrimidine, mA and mG indicates 2′-O-methylated purine.

FIG. 13. Aptamer tP19 conjugated to sticky end (SE) nucleic acidsequence with intermediate seven C3 carbon spacer, and predictedstructure. SE1-3—sticky end sequences and complementary sequences.

FIGS. 14A-14B. Nucleic acid sequence of components of bi-specificaptamer targeting one of mHSP70, vimentin or HSP90 and CCR5. Bi-specificaptamer is a non-covalent complex of one of the sequences depicted in(FIG. 14A) and the sequence depicted in (FIG. 14B). (FIG. 14A) Nucleicacid sequence of (i) full length P19 aptamer capable of binding tomHSP70 conjugated to a sticky sequence (bold) with intermediate seven C3carbon spacer (each C3 carbon represented by “o”), (ii) P1 aptamercapable of binding to mHSP70 conjugated to a sticky sequence (bold) withintermediate seven C3 carbon spacer (each C3 carbon represented by “o”),(iii) P15 aptamer capable of binding to vimentin conjugated to a stickysequence (bold) with intermediate seven C3 carbon spacer (each C3 carbonrepresented by “o”), (iv) P11 aptamer capable of binding to HSP90conjugated to a sticky sequence (bold) with intermediate seven C3 carbonspacer (each C3 carbon represented by “o”), (v) P7 aptamer capable ofbinding to HSP90 conjugated to a sticky sequence (bold) withintermediate seven C3 carbon spacer (each C3 carbon represented by “o”),(vi) P6 aptamer capable of binding to HSP90 conjugated to a stickysequence (bold) with intermediate seven C3 carbon spacer (each C3 carbonrepresented by “o”); (FIG. 14B) Nucleic acid sequence of CCR5 aptamerconjugated to a sticky sequence (bold) with intermediate five C3 carbonspacer (each C3 carbon represented by “o”). fC and fU indicates2′-fluoro pyrimidine, mA and mG indicates 2′-O-methylated purine.

FIG. 15. Sticky end (SE) sequences. A pair of complementary SE sequencesare used to form the bi-specific aptamer complex. SE 1 is conjugated tothe 3′ or 5′ end of one aptamer and one or SE2 or SE3 is conjugated tothe 3′ or 5′ end of the other aptamer. Aptamer-SE conjugates are mixedand allowed to form a bi-specific aptamer complex. SE1 [SEQ ID NO: 25].SE2 [SEQ ID NO: 26] complementary to SE1 3′-5′. SE3 [SEQ ID NO: 27]complementary to SE1 5′-3′.

FIG. 16. Diagram illustrating immunological synapse formed bybi-specific aptamers with a target cancer cell and T-cell.

FIGS. 17A-17D. Secondary structures and flow cytometry binding assay.(FIG. 17A) P15 was selected from randomized N40 RNA libraries. Thesecondary structure was predicted using the NUPACK software. (FIG. 17Band FIG. 17C) Cy3-labeled P19 and P1 aptamers (200 nM) were assessed forbinding efficiency by flow cytometry in PANC-1 and control Huh7 cells.The data show the percentage of positively stained cells from triplicateexperiments. The error bars represent the standard deviation (STD). Huh7CC (Huh7 unstained cell control), PANC-1 CC (PANC-1 unstained cellcontrol), Huh7 Lib (Huh 7 staining control with a Cy3-labeled library),PANC-1 Lib (PANC-1 staining control with a Cy3-labeled library), Huh7P15 (Huh7 stained with P15), PANC-1 P15 (PANC-1 stained with P15).Student's t test **: P<0.01. (FIG. 17D) The dissociation constant(K_(D)) was measured by flow cytometry using increasing concentrationsof Cy3-labeled aptamers (from 15.6 to 500 nM). The mean fluorescenceintensity (MFI) was measured and calculated using a one-site bindingmodel for non-linear regression.

FIG. 18. Fluorescence micrographs showing cell internalization. Thepancreatic cell lines PANC-1, AsPC-1, CFPAC-1, MIA PaCa-2 and BxPC-3were treated with 100 nM of the Cy3-labeled P15 aptamer and analyzed byconfocal microscopy. All of the pancreatic lines showed punctate regionsof Cy3 labeling. The Huh7 negative cells were also treated with 100 nMof Cy3-labeled P15 aptamers to show negative staining. Red: Cy3-labeledRNA. Blue: Hoechst 33342. Scale bar: 10 μm.

FIGS. 19A-19C. Tandem MS/MS spectra. (FIG. 19A) Polyacrylamide gelelectrophoresis (SDS-PAGE) was used to separate immobilized proteinsamples after pulldown with biotinylated P15 and irrelevant RNAs. Shownare Coomassie-stained gels M (Marker), total cell lysate (lane 1), P15(lane 2), NC (irrelevant RNA, lane 3). Arrow indicated the target. (FIG.19B) Peptide matching and MS/MS spectrum of P15 affinity-purifiedpeptides. Inset: Amino acid sequence of the parent peptide showing b-and y-ion series coverage. The target epitope was highlighted in yellow.Sequence: SEQ ID NO:40. (FIG. 19C) The aptamer-antibody competitionassay was employed to validate the target. The Cy3-labeled P15 aptamerwas used to compete with vimentin antibodies. The fluorescence intensitywas quantified (AU: arbitrary units). Student's t test *: P<0.05.

FIG. 20. Nucleic acid sequence of TfR aptamers.

FIG. 21. Nucleic acid sequence of PDGF-a aptamers.

FIG. 22. Schematic structure of bispecific aptamers for T cellengagement. Chemically synthesized CD3ε aptamer (CD3e2) and tP19 aptamerare non-covalently linked via complementary “sticky bridge” sequences tocreate a bispecific conjugate.

FIGS. 23A-23B. C3e2 (FIG. 23A) and C3e3 (FIG. 23B) both show multiplestem-loop structures.

FIGS. 24A-24B. Binding assay with CD3ε aptamer C3e2 and HEK cell linesstably expressing EGFP-CD3ε fusion proteins. (FIG. 24A) Aptamer C3e2binds HEK cells expressing human CD3ε. Visualization: Red channel(Cy3-labeled aptamer C3e2 (100 nM)); Green channel (EGFP-human CD3ε);Blue channel (Hoechst 33342). Scale bars: 10 μm. (FIG. 24B) Aptamer C3e2binds HEK cells expressing mouse CD3ε. Visualization: Red channel(Cy3-labeled aptamer C3e2 (100 nM)); Green channel (EGFP-mouse CD3ε);Blue channel (Hoechst 33342). Scale bars: 10 μm.

FIGS. 25A-25B. Binding assay with CD3ε aptamer C3e3 and HEK cell linesstably expressing EGFP-CD3ε fusion proteins. (FIG. 25A) Aptamer C3e3binds HEK cells expressing human CD3ε. Visualization: Red channel(Cy3-labeled aptamer C3e3 (100 nM)); Green channel (EGFP-human CD3ε);Blue channel (Hoechst 33342). Scale bars: 10 μm. (FIG. 25B) Aptamer C3e3binds HEK cells expressing mouse CD3ε. Visualization: Red channel(Cy3-labeled aptamer C3e3 (100 nM)); Green channel (EGFP-mouse CD3ε);Blue channel (Hoechst 33342). Scale bars: 10 μm.

FIGS. 26A-26B. Binding of CD3ε aptamers to human T cells. (FIG. 26A)Human T cells (1×10⁵ cells/mL) were incubated with 500 nM of Cy3-labeledaptamer C3e2. After washing, flow cytometry analysis was performed. Thehistogram shift indicates strong binding by C3e2. (FIG. 26B) Human Tcells (1×10⁵ cells/mL) were incubated with 500 nM of Cy3-labeled aptamerC3e3. After washing, flow cytometry analysis was performed. Thehistogram shift indicates strong binding by C3e3.

DETAILED DESCRIPTION OF THE INVENTION Definitions

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N Y 1989).

Any methods, devices and materials similar or equivalent to thosedescribed herein can be used in the practice of this invention. Thefollowing definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single-, double- or multiple-stranded form,or complements thereof. The term “polynucleotide” refers to a linearsequence of nucleotides. The term “nucleotide” typically refers to asingle unit of a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA (including siRNA),and hybrid molecules having mixtures of single and double stranded DNAand RNA. Nucleic acids can be linear or branched. For example, nucleicacids can be a linear chain of nucleotides or the nucleic acids can bebranched, e.g., such that the nucleic acids comprise one or more arms orbranches of nucleotides. Optionally, the branched nucleic acids arerepetitively branched to form higher ordered structures such asdendrimers and the like.

Nucleic acids, including nucleic acids with a phosphothioate backbonecan include one or more reactive moieties. As used herein, the termreactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,noncovalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

The terms also encompass nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphodiester derivativesincluding, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate(also known as phosphothioate), phosphorodithioate, phosphonocarboxylicacids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformicacid,methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, modified sugars, and non-ribosebackbones (e.g. phosphorodiamidate morpholino oligos or locked nucleicacids (LNA)), including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. In embodiments, the internucleotide linkages in DNAare phosphodiester, phosphodiester derivatives, or a combination ofboth.

The words “complementary” or “complementarity” refer to the ability of anucleic acid in a polynucleotide to form a base pair with anothernucleic acid in a second polynucleotide. For example, the sequence A-G-Tis complementary to the sequence T-C-A. Complementarity may be partial,in which only some of the nucleic acids match according to base pairing,or complete, where all the nucleic acids match according to basepairing.

The term “aptamer” as provided herein refers to oligonucleotides (e.g.short oligonucleotides or deoxyribonucleotides), that bind (e.g. withhigh affinity and specificity) to proteins, peptides, and smallmolecules. An aptamer may be referred to as an oligonucleotide basedtarget binding moiety. Aptamers may be RNA or DNA. Aptamers may havesecondary or tertiary structure and, thus, may be able to fold intodiverse and intricate molecular structures.

Aptamers can be selected in vitro from very large libraries ofrandomized sequences by the process of systemic evolution of ligands byexponential enrichment (SELEX as described in Ellington A D, Szostak J W(1990) In vitro selection of RNA molecules that bind specific ligands.Nature 346:818-822; Tuerk C, Gold L (1990) Systematic evolution ofligands by exponential enrichment: RNA ligands to bacteriophage T4 DNApolymerase. Science 249:505-510) or by developing SOMAmers (slowoff-rate modified aptamers) (Gold L et al. (2010) Aptamer-basedmultiplexed proteomic technology for biomarker discovery. PLoS ONE5(12):e15004). Applying the SELEX and the SOMAmer technology includesfor instance adding functional groups that mimic amino acid side chainsto expand the aptamer's chemical diversity. As a result high affinityaptamers for a protein may be enriched and identified. Aptamers mayexhibit many desirable properties for targeted drug delivery, such asease of selection and synthesis, high binding affinity and specificity,low immunogenicity, and versatile synthetic accessibility. Anticanceragents (e.g. chemotherapy drugs, toxins, and siRNAs) may be successfullydelivered to cancer cells in vitro using aptamers.

Aptamers are nucleic acid molecules characterised by the ability to bindto a target molecule with high specificity and high affinity. Almostevery aptamer identified to date is a non-naturally occurring molecule.

Aptamers may be DNA or RNA molecules and may be single stranded ordouble stranded. The aptamer may comprise chemically modifiednucleotides or nucleosides, for example in which the sugar and/orphosphate and/or base is chemically modified. Such modifications mayimprove the stability of the aptamer or make the aptamer more resistantto degradation. The aptamers of the present invention may includechemical modifications as described herein such as a chemicalsubstitution at a sugar position, a phosphate position, and/or a baseposition of the nucleic acid including, for example, incorporation of amodified nucleotide, incorporation of a capping moiety (e.g. 3′capping), conjugation to a high molecular weight, non-immunogeniccompound (e.g. polyethylene glycol (PEG)), conjugation to a lipophiliccompound, substitutions in the phosphate backbone. Base modificationsmay include 5-position pyrimidine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of5-bromo- or 5-iodo-uracil, backbone modifications. Sugar modificationsmay include 2′-amine nucleotides (2′-NH₂), 2′-fluoro nucleotides (2′-F),and 2′-O-methyl (2′-OMe) nucleotides. A wide range of nucleotide,nucleoside, base and phosphate modifications are known to those orordinary skill in the art, e.g. as described in Eaton et al., Bioorganic& Medicinal Chemistry, Vol. 5, No. 6, pp 1087-1096, 1997.

Aptamers may be synthesised by methods which are well known to theskilled person. For example, aptamers may be chemically synthesised,e.g. on a solid support. Solid phase synthesis may use phosphoramiditechemistry. Briefly, a solid supported nucleotide is detritylated, thencoupled with a suitably activated nucleoside phosphoramidite to form aphosphite triester linkage. Capping may then occur, followed byoxidation of the phosphite triester with an oxidant, typically iodine.The cycle may then be repeated to assemble the aptamer (e.g., see Sinha,N. D.; Biemat, J.; McManus, J.; Köster, H. Nucleic Acids Res. 1984, 12,4539; and Beaucage, S. L.; Lyer, R. P. (1992). Tetrahedron 48 (12):2223).

Aptamers can be thought of as the nucleic acid equivalent of monoclonalantibodies and often have K_(d)'s in the nM or pM range, e.g. less thanone of 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM. As withmonoclonal antibodies, they may be useful in virtually any situation inwhich target binding is required, including use in therapeutic anddiagnostic applications, in vitro or in vivo. In vitro diagnosticapplications may include use in detecting the presence or absence of atarget molecule.

Aptamers according to the present invention may be provided in purifiedor isolated form. Aptamers according to the present invention may beformulated as a pharmaceutical composition or medicament.

A “tumor cell antigen aptamer” is an aptamer that has high affinity andspecificity for a tumor cell antigen, as defined herein. WO2013/154735and FIG. 10 describe the pancreatic cancer cell binding aptamers P19,P15, P1, P11, P7, P6 and the consensus sequence SEQ ID NO:8. Thesequence of a truncated P19 aptamer is shown in FIG. 10.

In some embodiments the tumor cell antigen aptamer is a HSP70 bindingaptamer. HSP70 binding aptamers such as P19, tP19 and P1 are describedin WO2013/154735 and in U.S. provisional patent application No.62/141,156, incorporated herein by reference.

In some embodiments the tumor cell antigen aptamer is a vimentin bindingaptamer. Vimentin binding aptamers such as P15 are described inWO2013/154735, incorporated herein by reference.

In some embodiments the tumor cell antigen aptamer is a HSP90 bindingaptamer. HSP90 binding aptamers such as P11, P7 and P6 are described inWO2013/154735, incorporated herein by reference.

Pancreatic cancer cell binding aptamers, including HSP70, vimentin andHSP90 binding aptamers, include aptamers comprising a nucleic acidsequence according to one of SEQ ID NOs 1 to 8, or an aptamer having anucleic acid sequence having a degree of primary sequence identity of atleast 80% to one of SEQ ID NOs 1 to 8. In some embodiments a pancreaticcancer cell, HSP70, vimentin or HSP90 binding aptamer may have a nucleicacid sequence consisting of one of SEQ ID NOs 1 to 8. In a bi-specificaptamer according to the present invention the pancreatic cancer cell,HSP70, vimentin or HSP90 binding part, e.g. excluding any linker orspacer nucleic acid sequence or sticky bridge may have a nucleic acidsequence comprising or consisting of one of SEQ ID NOs 1 to 8 asdescribed above and herein.

In some embodiments the tumor cell antigen aptamer is a transferrinreceptor (TfR) binding aptamer. TfR binding aptamers such as TR14 andTR18 (shown in FIG. 20) are described in PCT/US15/55792, incorporatedherein by reference.

TfR binding aptamers include aptamers comprising a nucleic acid sequenceaccording to one of SEQ ID NOs 28 to 30, or an aptamer having a nucleicacid sequence having a degree of primary sequence identity of at least80% to one of SEQ ID NOs: 28 to 30. In some embodiments a TfR bindingaptamer may have a nucleic acid sequence consisting of one of SEQ IDNOs: 28 to 30. In a bi-specific aptamer according to the presentinvention the TfR binding part, e.g. excluding any linker or spacernucleic acid sequence or sticky bridge may have a nucleic acid sequencecomprising or consisting of one of SEQ ID Nos: 28 to 30 as describedabove and herein.

In some embodiments the tumor cell antigen aptamer is an alpha-typeplatelet-derived growth factor receptor (PDGFR-a) binding aptamer.PDGFR-a binding aptamers such as PDR3 and PDR9 (shown in FIG. 21) aredescribed in PCT/US15/55815, incorporated herein by reference.

PDGFR-a binding aptamers include aptamers comprising a nucleic acidsequence according to one of SEQ ID NOs: 31 and 32, or an aptamer havinga nucleic acid sequence having a degree of primary sequence identity ofat least 80% to one of SEQ ID NOs: 31 and 32. In some embodiments aPDGFR-a binding aptamer may have a nucleic acid sequence consisting ofone of SEQ ID NOs: 31 and 32. In a bi-specific aptamer according to thepresent invention the PDGFR-a binding part, e.g. excluding any linker orspacer nucleic acid sequence or sticky bridge may have a nucleic acidsequence comprising or consisting of one of SEQ ID NOs: 31 and 32 asdescribed above and herein.

An “immune cell surface protein aptamer” is an aptamer that has highaffinity and specificity for an immune cell surface protein, as definedherein.

In some embodiments the immune cell surface protein aptamer is a CCR5binding aptamer. CCR5 binding aptamers are described in Zhou et al.,2015, Chemistry & Biology 22, 379-390 Mar. 19, 2015 and in co-pendingU.S. patent application Ser. No. 14/801,710, each specificallyincorporated herein by reference. CCR5 binding aptamers include aptamerscomprising a nucleic acid sequence according to one of SEQ ID NOs 9 to16, or an aptamer having a nucleic acid sequence having a degree ofprimary sequence identity of at least 80% to one of SEQ ID NOs 9 to 16.In some embodiments a CCR5 binding aptamer may have a nucleic acidsequence consisting of one of SEQ ID NOs 9 to 16. In some embodimentseach pyrimidine is a 2′fluoropyrimidine. In a bi-specific aptameraccording to the present invention the CCR5 binding part, e.g. excludingany linker or spacer nucleic acid sequence or sticky bridge may have anucleic acid sequence comprising or consisting of one of SEQ ID NOs 9 to16 as described above and herein.

In some embodiments the immune cell surface protein aptamer is a CCR7binding aptamer.

In some embodiments the immune cell surface protein aptamer is a CD2binding aptamer.

In some embodiments the immune cell surface protein aptamer is a CD3binding aptamer. CD3 binding aptamers include aptamers comprising anucleic acid sequence according to one of SEQ ID NOs 37 and 38, or anaptamer having a nucleic acid sequence having a degree of primarysequence identity of at least 80% to one of SEQ ID NOs 37 and 38. Insome embodiments a CD3 binding aptamer may have a nucleic acid sequenceconsisting of one of SEQ ID NOs 37 and 38. In some embodiments eachpyrimidine is a 2′fluoropyrimidine. In a bi-specific aptamer accordingto the present invention the CD3 binding part, e.g. excluding any linkeror spacer nucleic acid sequence or sticky bridge may have a nucleic acidsequence comprising or consisting of one of SEQ ID NOs 37 and 38 asdescribed above and herein. In embodiment, a CD3 binding aptamerincludes an aptamer comprising a nucleic acid sequence of SEQ ID NO: 34or 35, including a linker or spacer nucleic acid sequence or stickybridge.

In some embodiments the immune cell surface protein aptamer is a CD4binding aptamer. CD4 binding aptamers are described in Zhou, Qing et al.“Aptamer-Containing Surfaces for Selective Capture of CD4 ExpressingCells.” Langmuir: the ACS journal of surfaces and colloids 28.34 (2012):12544-12549. PMC. Web. 8 Feb. 2016; Zhang et al., American Journal ofClinical Pathology, Volume 134, Issue 4, 1 Oct. 2010; Wheeler et al., JClin Invest. 2011; 121(6):2401-2412, each specifically incorporatedherein by reference.

In some embodiments the immune cell surface protein aptamer is a CD7binding aptamer. CD7 binding aptamers are described in WO2014/147559,specifically incorporated herein by reference.

In some embodiments the immune cell surface protein aptamer is a CD8binding aptamer. CD8 binding aptamers are described in Wang et al., JAllergy Clin Immunol. 2013 September; 132(3):713-722; Oelkrug, C., Sack,U., Boldt, A., Nascimento, I. C., Ulrich, H. and Fricke, S. (2015),Antibody- and aptamer-strategies for GVHD prevention. Journal ofCellular and Molecular Medicine, 19: 11-20, each specificallyincorporated herein by reference.

In some embodiments the immune cell surface protein aptamer is a PD-1binding aptamer. PD-1 binding aptamers are described in, Prodeus et alMolecular Therapy Nucleic Acids (2015) 4 e237, Ti-Hsuan Ku Sensors 2015,15, 16281-16313, and WO2016/019270, each specifically incorporatedherein by reference.

In some embodiments the immune cell surface protein aptamer is a CTLA4binding aptamer. CTLA4 binding aptamers are described in Herrmann etal., J Clin Invest. 2014; 124(7):2977-2987, Gilboa et al., Clin CancerRes; 19(5); 1054-62, and Santulli-Marotto et al., Cancer Res. 2003 Nov.1; 63(21):7483-9, each specifically incorporated herein by reference.

Aptamers are normally mono-specific, i.e. having high affinity andspecificity for a single target molecule.

The nucleic acid sequence of a mono-specific aptamer, or mono-specificpart of a bi-specific aptamer, according to the present invention mayoptionally have a minimum length of one of 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides

The nucleic acid sequence of a mono-specific aptamer, or mono-specificpart of a bi-specific aptamer, according to the present invention mayoptionally have a maximum length of one of 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 nucleotides.

The nucleic acid sequence of a mono-specific aptamer, or mono-specificpart of a bi-specific aptamer, according to the present invention mayoptionally have a length of one of 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

The nucleic acid sequence of a mono-specific aptamer or mono-specificpart of a bi-specific aptamer (including when present in a bi-specificaptamer complex), according to the present invention may have a degreeof primary sequence identity with one of SEQ ID NOs 1 to 24 or 28 to 32,that is at least one of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

A “bi-specific aptamer” is an aptamer based compound or composition thathas high affinity and specificity for two, or at least two, differenttarget molecules. A bi-specific aptamer may be comprised of the nucleicacid sequence of two mono-specific aptamers. A bi-specific aptamer maybe a complex or conjugate of two mono-specific aptamers. The nucleicacid sequences of the two mono-specific aptamers may be brought togetherto form a complex, which may be a covalent or non-covalent complex. Insome embodiments the bi-specific aptamer may comprise the nucleic acidsequence of a tumor cell antigen aptamer in complex with the nucleicacid sequence of an immune cell surface protein aptamer.

As such, a bi-specific aptamer may be a complex of a tumor cell antigenbinding moiety and an immune cell surface protein binding moiety.

A covalent complex may be provided by forming a covalent bond betweenmembers of the complex. In some embodiments a bi-specific aptamer may beformed by synthesizing a single oligonucleotide molecule that comprisesthe nucleic acid sequence of a first mono-specific aptamer followed bythe nucleic acid sequence of a second mono-specific aptamer, optionallywith a linker between the two sequences. The linker may comprise one ormore of an oligonucleotide sequence, hydrocarbon spacer elements such asoptionally substituted C₁₋₃₀ alkyl or optionally substituted C₂₋₃₀alkenyl; or polyethylene glycol molecule(s). In some embodiments thelinker may be a polycarbon linker, consistent with formation of a“sticky bridge”. The polycarbon linker may be an optionally substitutedC₁₀₋₃₀ alkyl, optionally substituted C₁₀₋₁₅ alkyl, optionallysubstituted C₁₅₋₂₀ alkyl, optionally substituted C₂₀₋₂₅ alkyl,optionally substituted C₂₅₋₃₀ alkyl, optionally substituted C₁₀₋₃₀alkenyl, optionally substituted C₁₀₋₁₅ alkenyl, optionally substitutedC₁₅₋₂₀ alkenyl, optionally substituted C₂₀₋₂₅ alkenyl, optionallysubstituted C₂₅₋₃₀ alkenyl.

A non-covalent complex may be provided by forming one or morenon-covalent bonds between members of the complex. Non-covalentcomplexes may be maintained by hydrogen bonding, van der Waal forces andoptionally ionic interaction. In some embodiments a bi-specific aptamermay be formed by attaching one of a pair of linker moieties to thenucleic acid sequence of each of two mono-specific aptamers, where thelinker moieties have affinity or complementarity for each other, andallowing the linker moieties to bind and form a non-covalent complex.Examples of suitable linker moieties include a pair of single strandedoligonucleotides having complementary sequences that permithybridization or tag and capture element pairs such as biotin andavidin/strepavidin.

As used herein, the term “conjugate,” “bioconjugate” or “bioconjugatereactive group” or “bioconjugate linker” refers to the associationbetween atoms or molecules. The association can be direct or indirect.For example, a conjugate between a first moiety (e.g. —NH₂, —COOH,—N-hydroxysuccinimide, or -maleimide) and a second moiety (e.g.,sulfhydryl, sulfur-containing amino acid) provided herein can be direct,e.g., by covalent bond or linker, or indirect, e.g., by non-covalentbond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond,halogen bond), van der Waals interactions (e.g. dipole-dipole,dipole-induced dipole, London dispersion), ring stacking (pi effects),hydrophobic interactions and the like). In embodiments, conjugates areformed using conjugate chemistry including, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982. In embodiments, thefirst moiety (e.g., a tumor cell antigen binding moiety) isnon-covalently attached to the second moiety on the immune cell surfaceprotein binding moiety through a non-covalent chemical linker orcovalent chemical linker formed by a reaction between a component of thefirst moiety and a component of the second moiety. In embodiments, thefirst moiety (e.g., a tumor cell antigen binding moiety) includes one ormore reactive moieties, e.g., a covalent reactive moiety, as describedherein (e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide,maleimide or thiol reactive moiety). In embodiments, the first moiety(e.g., a tumor cell antigen binding moiety) includes a linker (e.g.,first linker) with one or more reactive moieties, e.g., a covalentreactive moiety, as described herein (e.g., alkyne, azide, amine, ester,N-hydroxy-succinimide, maleimide or thiol reactive moiety). Inembodiments, the second moiety (e.g., an immune cell surface proteinbinding moiety) includes one or more reactive moieties, e.g., a covalentreactive moiety, as described herein (e.g., alkyne, azide, amine, ester,N-hydroxy-succinimide, maleimide or thiol reactive moiety). Inembodiments, the second moiety (e.g., an immune cell surface proteinbinding moiety) includes a linker with one or more reactive moieties,e.g., a covalent reactive moiety, as described herein (e.g., alkyne,azide, amine, ester, N-hydroxy-succinimide, maleimide or thiol reactivemoiety).

Useful reactive moieties or functional groups used for conjugatechemistries herein include, for example: (a) carboxyl groups and variousderivatives thereof including, but not limited to, Nhydroxysuccinimideesters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles,thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromaticesters; (b) hydroxyl groups which can be converted to esters, ethers,aldehydes, etc. (c) halo alkyl groups wherein the halide can be laterdisplaced with a nucleophilic group such as, for example, an amine, acarboxylate anion, thiol anion, carbanion, or an alkoxide ion, therebyresulting in the covalent attachment of a new group at the site of thehalogen atom; (d) dienophile groups which are capable of participatingin Diels-Alder reactions such as, for example, maleimido groups; (e)aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition; (f) sulfonyl halide groupsfor subsequent reaction with amines, for example, to form sulfonamides;(g) thiol groups, which can be converted to disulfides, reacted withacyl halides, or bonded to metals such as gold; (h) amine or sulfhydrylgroups, which can be, for example, acylated, alkylated or oxidized; (i)alkenes, which can undergo, for example, cycloadditions, acylation,Michael addition, etc; (j) epoxides, which can react with, for example,amines and hydroxyl compounds; (k) phosphoramidites and other standardfunctional groups useful in nucleic acid synthesis; (l) metal siliconoxide bonding; (m) metal bonding to reactive phosphorus groups (e.g.phosphines) to form, for example, phosphate diester bonds; and (n)sulfones, for example, vinyl sulfone.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the chemical stability of theproteins described herein. By way of example, the nucleic acids caninclude a vinyl sulfone or other reactive moiety. Optionally, thenucleic acids can include a reactive moiety having the formula S—S—R. Rcan be, for example, a protecting group. Optionally, R is hexanol. Asused herein, the term hexanol includes compounds with the formulaC6H130H and includes, 1-hexanol, 2-hexanol, 3-hexanol,2-methyl-1-pentanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol,2-methyl-2-pentanol, 3-methyl-2-pentanol, 4-methyl-2-pentanol,2-methyl-3-pentanol, 3-methyl-3-pentanol, 2,2-dimethyl-1-butanol,2,3-dimethyl-1-butanol, 3,3-dimethyl-1-butanol, 2,3-dimethyl-2-butanol,3,3-dimethyl-2-butanol, and 2-ethyl-1-butanol. Optionally, R is1-hexanol.

In embodiments, oligonucleotides, including aptamers, antisense nucleicacids etc, may be formed into a non-covalent complex through the use ofa “sticky bridge”. A sticky bridge comprises an oligonucleotide (“stickysequence” or “sticky end”) positioned at the 3′- or 5′-end of a firstaptamer oligonucleotide sequence. A complementary oligonucleotide(“sticky sequence” or “sticky end”) is positioned at the 3′- or 5′-endof a second, preferably different, aptamer oligonucleotide sequence. Thecomplementary sticky sequences are allowed to hybridise and form anon-covalent complex comprising the first and second aptamers. Thesticky sequence may be GC or AU rich, and each sticky sequence maycomprise about 16 nucleotides, e.g. 14 to 20 nucleotides or one of 14,15, 16, 17, 18, 19 or 20 nucleotides. Examples of complementary pairs ofsticky sequences are SEQ ID NOs: 25 and 26 or SEQ ID NOs: 25 and 27(FIG. 15).

A polycarbon linker may be incorporated between the sticky sequence andaptamer oligonucleotide sequence. The polycarbon linker providesrigidity to the aptamer, decreasing the likelihood that the inclusion ofthe additional sticky sequences will interfere with proper aptamerfolding (e.g. see Zhou J, Swiderski P, Li H, et al. Selection,characterization and application of new RNA HIV gp 120 aptamers forfacile delivery of Dicer substrate siRNAs into HIV infected cells.Nucleic Acids Res. 2009; 37(9):3094-3109).

The polycarbon linker may comprise one or more of an oligonucleotidesequence, hydrocarbon spacer elements such as optionally substitutedC₁₋₃₀ alkyl or optionally substituted C₂₋₃₀ alkenyl; or polyethyleneglycol molecule(s). In some embodiments the polycarbon linker may be apolycarbon linker, consistent with formation of a “sticky bridge”. Thepolycarbon linker may be an optionally substituted C₁₀₋₃₀ alkyl,optionally substituted C₁₀₋₁₅ alkyl, optionally substituted C₁₅₋₂₀alkyl, optionally substituted C₂₀₋₂₅ alkyl, optionally substitutedC₂₅₋₃₀ alkyl, optionally substituted C₁₀₋₃₀ alkenyl, optionallysubstituted C₁₀₋₁₅ alkenyl, optionally substituted C₁₅₋₂₀ alkenyl,optionally substituted C₂₀₋₂₅ alkenyl, optionally substituted C₂₅₋₃₀alkenyl.

Accordingly, a bispecific aptamer according to the present invention maycomprise a first aptamer component, e.g. comprising one of SEQ ID NOs 1to 8, 28 to 32, covalently conjugated at the 3′ or 5′ end to a stickyend sequence, e.g. one of SEQ ID NOs 25 to 27, optionally via apolycarbon linker spacer, complexed with a second aptamer component,e.g. comprising one of SEQ ID NOs 9 to 16, covalently conjugated at the3′ or 5′ end to a complementary sticky end sequence, e.g. one of SEQ IDNOs 25 to 27, optionally via a polycarbon linker spacer.

The length of a bi-specific aptamer will reflect the length of thenucleic acid sequence of each mono-specific aptamer incorporated in thebi-specific aptamer, and optionally the length of any linker that isincluded.

A bi-specific aptamer may therefore be defined as a complex of a firstmono-specific aptamer having a defined length and degree of primarysequence identity to one of SEQ ID NOs: 1 to 8 or 28 to 32 and a secondmono-specific aptamer having a defined length and degree of primarysequence identity to one of SEQ ID NOs: 9 to 16. In embodiments thefirst mono-specific aptamer is a tumor cell antigen aptamer and thesecond mono-specific aptamer is an immune cell surface protein aptamer.

An “antisense nucleic acid” as referred to herein is a nucleic acid(e.g. DNA or RNA molecule) that is complementary to at least a portionof a specific target nucleic acid (e.g. an mRNA translatable into aprotein) and is capable of reducing transcription of the target nucleicacid (e.g. mRNA from DNA) or reducing the translation of the targetnucleic acid (e.g. mRNA) or altering transcript splicing (e.g. singlestranded morpholino oligo). See, e.g., Weintraub, Scientific American,262:40 (1990). Typically, synthetic antisense nucleic acids (e.g.oligonucleotides) are generally between 15 and 25 bases in length. Thus,antisense nucleic acids are capable of hybridizing to (e.g. selectivelyhybridizing to) a target nucleic acid (e.g. target mRNA). Inembodiments, the antisense nucleic acid hybridizes to the target nucleicacid sequence (e.g. mRNA) under stringent hybridization conditions. Inembodiments, the antisense nucleic acid hybridizes to the target nucleicacid (e.g. mRNA) under moderately stringent hybridization conditions.Antisense nucleic acids may comprise naturally occurring nucleotides ormodified nucleotides such as, e.g., phosphorothioate, methylphosphonate,and -anomeric sugar-phosphate, backbone modified nucleotides.

In the cell, the antisense nucleic acids hybridize to the correspondingmRNA, forming a double-stranded molecule. The antisense nucleic acidsinterfere with the translation of the mRNA, since the cell will nottranslate an mRNA that is double-stranded. The use of antisense methodsto inhibit the in vitro translation of genes is well known in the art(Marcus-Sakura, Anal. Biochem., 172:289, (1988)). Further, antisensemolecules which bind directly to the DNA may be used. Antisense nucleicacids may be single or double stranded nucleic acids. Non-limitingexamples of antisense nucleic acids include siRNAs (including theirderivatives or pre-cursors, such as nucleotide analogs), short hairpinRNAs (shRNA), micro RNAs (miRNA), saRNAs (small activating RNAs) andsmall nucleolar RNAs (snoRNA) or certain of their derivatives orprecursors.

A “siRNA,” “small interfering RNA,” “small RNA,” or “RNAi” as providedherein, refers to a nucleic acid that forms a double stranded RNA, whichdouble stranded RNA has the ability to reduce or inhibit expression of agene or target gene when present in the same cell as the gene or targetgene. The complementary portions of the nucleic acid that hybridize toform the double stranded molecule typically have substantial or completeidentity. In one embodiment, a siRNA or RNAi is a nucleic acid that hassubstantial or complete identity to a target gene and forms a doublestranded siRNA. In embodiments, the siRNA inhibits gene expression byinteracting with a complementary cellular rnRNA thereby interfering withthe expression of the complementary mRNA. Typically, the nucleic acid isat least about 15-50 nucleotides in length (e.g., each complementarysequence of the double stranded siRNA is 15-50 nucleotides in length,and the double stranded siRNA is about 15-50 base pairs in length). Inother embodiments, the length is 20-30 base nucleotides, preferablyabout 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

A “saRNA,” or “small activating RNA” as provided herein refers to anucleic acid that forms a double stranded RNA, which double stranded RNAhas the ability to increase or activate expression of a gene or targetgene when present in the same cell as the gene or target gene. Thecomplementary portions of the nucleic acid that hybridize to form thedouble stranded molecule typically have substantial or completeidentity. In one embodiment, a saRNA is a nucleic acid that hassubstantial or complete identity to a target gene and forms a doublestranded saRNA. Typically, the nucleic acid is at least about 15-50nucleotides in length (e.g., each complementary sequence of the doublestranded saRNA is 15-50 nucleotides in length, and the double strandedsaRNA is about 15-50 base pairs in length). In other embodiments, thelength is 20-30 base nucleotides, preferably about 20-25 or about 24-29nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleotides in length.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It can be,for example, in a homogeneous state and may be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified.

The term “purified” denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. In some embodiments, thenucleic acid or protein is at least 50% pure, optionally at least 65%pure, optionally at least 75% pure, optionally at least 85% pure,optionally at least 95% pure, and optionally at least 99% pure.

The term “isolated” may also refer to a cell or sample cells. Anisolated cell or sample cells are a single cell type that issubstantially free of many of the components which normally accompanythe cells when they are in their native state or when they are initiallyremoved from their native state. In certain embodiments, an isolatedcell sample retains those components from its natural state that arerequired to maintain the cell in a desired state. In some embodiments,an isolated (e.g. purified, separated) cell or isolated cells are cellsthat are substantially the only cell type in a sample. A purified cellsample may contain at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% of one type of cell. An isolated cell sample may beobtained through the use of a cell marker or a combination of cellmarkers, either of which is unique to one cell type in an unpurifiedcell sample. In some embodiments, the cells are isolated through the useof a cell sorter. In some embodiments, antibodies against cell proteinsare used to isolate cells.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://wW′.v.ncbi.nlm.nih.gov/BLAST/or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the compliment of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol 215:403-410(1990), respectively.

For specific proteins described herein (e.g., mHSP70), the named proteinincludes any of the protein's naturally occurring forms, variants orhomologs (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%or 100% activity compared to the native protein). In some embodiments,variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring form. In other embodiments, theprotein is the protein as identified by its NCBI sequence reference. Inother embodiments, the protein is the protein as identified by its NCBIsequence reference, homolog or functional fragment thereof.

The term “tumor cell antigen” refers to any protein, carbohydrate orother component that is abnormally expressed by a tumor cell or isexpressed by a tumor cell with an abnormal structure. A tumor cellantigen may be expressed at the cell surface by tumor/cancer cells ofthe tumor/cancer concerned. A tumor cell antigen may optionally becapable of eliciting an immune response. A tumor cell antigen may be aprotein, carbohydrate or other component that is normally expressedinside the cell, but is expressed at the cell surface or in/at the cellmembrane of a tumor cell.

A tumor cell antigen may be a tumor-specific antigen. Abnormalexpression of a tumor specific antigen may be associated with the causeof the cancer. Tumor specific antigens may be preferentially expressedon cells of the tumor/cancer and not on healthy cells of the same type.Accordingly, tumor cell antigens may be products of mutated oncogenes ortumor suppressor genes.

A tumor cell antigen may be a tumor-associated antigen. Tumor-associatedantigens may also be abnormally by the cell type concerned.Tumor-associated antigens are not normally associated with the cause ofthe cancer, their abnormal expression normally being associated with, ora consequence of, the cancer. Accordingly, tumor-associated antigens maybe products of overexpressed cellular proteins, tumor antigens producedby oncogenic viruses, oncofetal antigens, cell surface glycolipids orglycoproteins.

Tumor antigens are reviewed by Zarour H M, DeLeo A, Finn O J, et al.Categories of Tumor Antigens. In: Kufe D W, Pollock R E, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton(ON): BC Decker; 2003. Tumor cell antigens include oncofetal antigens:CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPVE6 and E7; overexpressed proteins: BING-4, calcium-activated chloridechannel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, Her2/neu, telomerase,mesothelin, SAP-1, surviving; cancer-testis antigens: BAGE, CAGE, GAGE,MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; lineage restrictedantigens: MART1, Gp100, tyrosinase, TRP-1/2, MC1R, prostate specificantigen; mutated antigens: β-catenin, BRCA1/2, CDK4, CML66, Fibronectin,MART-2, p53, Ras, TGF-βRII; post-translationally altered antigens: MUC1,idiotypic antigens: Ig, TCR. Other tumor cell antigens includeheat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90),glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinarpancreatic protein (FAPP), alkaline phosphatase placental-like 2(ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), proteintyrosine kinase 7 (PTK7), cyclophilin B.

For example, where the tumor cell is a breast cancer cell, the antigenmay be one of EpCAM (epithelial cell adhesion molecule), Her2/neu (HumanEpidermal growth factor Receptor 2), MUC-1, EGFR (epidermal growthfactor receptor), TAG-12 (tumor associated glycoprotein 12), IGF1 R(insulin-like growth factor 1 receptor), TACSTD2 (tumor associatedcalcium signal transducer 2), CD318, CD340, CD104, or N-cadherin.

For example, where the tumor cell is a prostate cancer cell, the antigenmay be one of EpCAM, MUC-1, EGFR, PSMA (prostate specific membraneantigen), PSA (prostate specific antigen), TACSTD2, PSCA (prostate stemcell antigen), PCSA (prostate cell surface antigen), CD318, CD104, orN-cadherin.

For example, where the tumor cell is a colorectal cancer cell, theantigen may be one of EpCAM, CD66c, CD66e, CEA (carcinoembryonicantigen), TACSTD2, CK20 (cytokeratin 20), CD104, MUC-1, CD318, orN-cadherin.

For example, where the tumor cell is a lung cancer cell the antigen maybe one or CK18, CK19, CEA, EGFR, TACSTD2, CD318, CD1 04, or EpCAM.

For example, where the tumor cell is a pancreatic cancer cell theantigen may be one of HSP70, mHSP70, vimentin, HSP90, MUC-1, TACSTD2,CEA, CD104, CD318, N-cadherin, or EpCAM1.

For example, where the tumor cell is an ovarian cancer cell the antigenmay be one of MUC-1, TACSTD2, CD318, CD104, N-cadherin, or EpCAM.

For example, where the tumor cell is a bladder cancer cell, the antigenmay be one of CD34, CD146, CD62, CD105, CD106, VEGF receptor (vascularendothelial growth factor receptor), MUC-1, TACSTD2, EpCAM, CD318, EGFR,6B5 or Folate binding receptor.

For example, where the tumor cell is a cancer stem cell, the antigen maybe one of CD133, CD135, CD 117, or CD34.

For example, where the tumor cell is a melanoma cancer cell, the antigenmay be one of the melanocyte differentiation antigens, oncofetalantigens, tumor specific antigens, SEREX antigens or a combinationthereof. Examples of melanocyte differentiation antigens, include butare not limited to tyrosinase, gp75, gp100, MART 1 or TRP-2. Examples ofoncofetal antigens include antigens in the MAGE family (MAGE-A1,MAGE-A4), BAGE family, GAGE family or NY-ESO1. Examples oftumor-specific antigens include CDK4 and 13-catenin. Examples of SEREXantigens include D-1 and SSX-2.

In some preferred embodiments the tumor cell antigen is HSP70 or mHSP70.In embodiments, the tumor cell is a pancreatic cancer cell. Inembodiments, the tumor cell is a glioblastoma cell. In embodiments, thetumor cell is a colon cancer cell.

The term “HSP70” refers to the family of approximately 70 kilodaltonheat shock proteins as well-known in the art. In some preferredembodiments, the HSP70 is mHSP70. The term “mHSP70” as provided hereinincludes any of the mitochondrial HSP70 (mHSP70) protein naturallyoccurring forms, homologs or variants that maintain the activity ofmHSP70 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to the native protein). In some embodiments,variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring form. In embodiments, the mHSP70protein is the protein as identified by the NCBI sequence referenceNP_004125.3 GI:24234688, homolog or functional fragment thereof mHSP70may also be referred to herein as mortalin, CSA, GRP-75, GRP75,HEL-S-124m, HSPA9B, MOT, MOT2, MTHSP75 or PBP74. Overexpression of HSP70in cancer is linked to poor prognosis, e.g. see Bagatell and Whitesell.,Mol Cancer Ther August 2004 3; 1021.

In some preferred embodiments the tumor cell antigen is vimentin. Inembodiments, the tumor cell is a pancreatic cancer cell. In embodiments,the tumor cell is a glioblastoma cell. In embodiments, the tumor cell isa colon cancer cell.

The term “vimentin” refers to the family of class III intermediatefilaments found in a number of health non-epithelial cells, includingmesenchymal stem cells. The term “vimentin” as provided herein includesany of the protein naturally occurring forms, homologs or variants thatmaintain the activity of vimentin (e.g., within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to the nativeprotein). In some embodiments, variants or homologs have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring form.In embodiments, the vimentin protein is the protein as identified by theUniProt accession no. P08670 NCBI sequence reference or by Genbankaccession no. NP_003371.2 GI:62414289. Vimentin is overexpressed invarious epithelial cancers, including prostate cancer, gastrointestinaltumors, tumors of the central nervous system, breast cancer, malignantmelanoma, and lung cancer (Satelli et al., Cell mol Life Sci 2011September; 68(18):3033-46.

In some preferred embodiments the tumor cell antigen is HSP90. Inembodiments, the tumor cell is a pancreatic cancer cell. In embodiments,the tumor cell is a glioblastoma cell. In embodiments, the tumor cell isa colon cancer cell.

The term “HSP90” refers to the family of approximately 90 kilodaltonheat shock proteins as well-known in the art. The term “HSP90” asprovided herein includes any of the protein naturally occurring forms,homologs or variants that maintain the activity of HSP90 (e.g., withinat least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to the native protein). In some embodiments, variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring form. In embodiments, the HSP90 protein is theprotein as identified by Genbank accession no. NP_001017963.2GI:153792590 or NP_005339.3 GI:154146191. Overexpression of HSP90 incancer is linked to poor prognosis, e.g. see Bagatell and Whitesell.,Mol Cancer Ther August 2004 3; 1021.

In some preferred embodiments the tumor cell antigen is transferrinreceptor.

The term “TfR” as provided herein includes any of the transferrinreceptor (TfR) protein naturally occurring forms, homologs or variantsthat maintain the activity of TfR (e.g., within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to the nativeprotein). In some embodiments, variants or homologs have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring form.In embodiments, the TfR protein is the protein as identified by the NCBIsequence reference NP_003225.2 GI: 189458817. In embodiments, the TfRprotein is the protein as identified by the NCBI sequence reference GI:189458816. In embodiments, the TfR protein is the protein as identifiedby the NCBI sequence reference GI: 189458818. In embodiments, the TfRprotein is the protein as identified by the NCBI sequence reference GI:189458817, homolog or functional fragment thereof. In embodiments, theTfR protein is the protein as identified by the NCBI sequence referenceGI: 189458816, homolog or functional fragment thereof. In embodiments,the TfR protein is the protein as identified by the NCBI sequencereference GI: 189458818, homolog or functional fragment thereof. Inembodiments, the TfR protein is encoded by a nucleic acid sequencecorresponding to Gene ID: 7037.

The transferrin receptor (TfR) is a membrane glycoprotein expressed onthe cellular surface and mediates cellular uptake of iron from theplasma glycoprotein transferrin. Iron uptake from transferrin involvesthe binding of transferrin to TfR. The bound transferrin is theninternalized through receptor-mediated endocytosis in an endocyticvesicle. The release of transferrin from TfR is induced by a decrease inthe pH within the endocytic vescile. TfR is expressed on a broad varietyof cells at varying levels. For example, TfR is highly expressed onimmature erythroid cells, placental tissue, and rapidly dividing cells,both normal and malignant. Thus, compounds capable of binding to TfR onthe surface of TfR-expressing cells and internalizing into the cellwould be very useful for targeted delivery of such compounds.

In some preferred embodiments the tumor cell antigen is alpha-typeplatelet-derived growth factor receptor.

The term “PDGFR-a” as provided herein includes any of the alpha-typeplatelet-derived growth factor receptor (PDGFR-a) protein naturallyoccurring forms, homologs or variants that maintain the tyrosine kinaseactivity of PDGFR-a (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native protein). In someembodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous aminoacid portion) compared to a naturally occurring form. In embodiments,the PDGFR-a protein is the protein as identified by the NCBI sequencereference NP_006197.1 GI:5453870. In embodiments, the PDGFR-a protein isthe protein as identified by the NCBI sequence reference GI:5453870,homolog or functional fragment thereof. In embodiments, the PDGFR-aprotein is encoded by a nucleic acid sequence corresponding to Gene ID:GI: 172072625.

Platelet-derived growth factor receptor alpha (PDGFR-a) is acell-surface tyrosine kinase receptor implicated in regulating cellproliferation, cellular differentiation, cell growth and development.PDGFR-a is frequently expressed by tumor cells, predominantly bymalignant tumor cells. The expression levels of PDGFR-a correlates withtumor growth, invasiveness, drug resistance and poor clinical outcomes.For example, PDGFR-a is highly over expressed in glioblastoma (GBM).Thus, compounds capable of binding to PDGFR on the surface ofPDGFR-expressing cells and internalizing into the cell are highlydesirable.

The term “immune cell surface protein” refers to any protein present atthe cell surface of an immune cell. In some embodiments it is a T-cellsurface protein, receptor or co-receptor. In some embodiments it is acluster of differentiation (CD) cell surface molecule, preferably whenpresent on an immune cell. In some embodiments it is a CC chemokinereceptor (CCR), preferably when present on an immune cell. In someembodiments it is a CXC chemokine receptor (CXCR), preferably whenpresent on an immune cell.

An “immune cell” may be a lymphocyte, white blood cell, T-cell(thymocyte), T-helper cell, cytotoxic T-cell, CD8+ T-cell, CD4+ T-cell,memory T-cell, suppressor T-cell, natural killer T-cell, gamma deltaT-cell, B cell, natural killer cell, leukocyte, macrophage, neutrophil,dendritic cell. An immune cell may express any one of CCR5, CCR7, CD2,CD3, CD4, CD7, CD8, PD-1, CTLA4 at the cell surface or in/at the cellmembrane. In some preferred embodiments an immune cell may be a T-cell,e.g. expressing a T-cell receptor on its surface. In some preferredembodiments the immune cell may be a CD8+ T-cell. In some preferredembodiments the immune cell may be a CD4+ T-cell. In some preferredembodiments the immune cell is a cytotoxic T-cell.

In preferred embodiments, the immune cell surface protein is selectedfrom the group consisting of CCR5, CCR7, CD2, CD3, CD4, CD7, CD8, PD-1,CTLA4.

The term “CCR5” as provided herein includes any of the C-C chemokinereceptor type 5 (CCR5) protein naturally occurring forms, homologs orvariants that maintain the activity of CCR5 (e.g., within at least 50%,80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to thenative protein). In some embodiments, variants or homologs have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurringform. In embodiments, the CCR5 protein is the protein as identified bythe UniProt sequence reference P51681. In embodiments, the CCR5 proteinis the protein as identified by the NCBI sequence reference NP_000570.1GI:4502639, homolog or functional fragment thereof. In embodiments, theCCR5 protein is encoded by a nucleic acid sequence corresponding to GeneID: GI:154091329.

Human CCR5 (C-C chemokine receptor type 5), a 7 pass transmembranereceptor expressed by T-cells and macrophages, serves as a co-receptorfor macrophage-tropic HIV-1. A loss of CCR5 is associated withresistance to HIV-1. Thus, CCR5 is an important co-receptor formacrophage-tropic virus, including HIV-I RS isolates. Variations in CCR5are associated with resistance or susceptibility to HIV-1. As anessential factor for viral entry, CCR5 has represented an attractivecellular target for the treatment of HIV-1. CCR5 is also known as CD195.

The term “CCR7” refers to C-C chemokine receptor type 7 (also calledCD197), well-known in the art. The term “CCR7” as provided hereinincludes any of the naturally occurring forms, homologs or variants thatmaintain the activity of CCR7 (e.g., within at least 50%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100% activity compared to the native protein). Insome embodiments, variants or homologs have at least 90%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity across the whole sequenceor a portion of the sequence (e.g. a 50, 100, 150 or 200 continuousamino acid portion) compared to a naturally occurring form. Inembodiments, the CCR7 protein is the protein as identified by the NCBIsequence reference NP_001288643.1 GI:683523912, homolog or functionalfragment thereof. CCR7 is a member of the G protein coupled receptorfamily.

The term “CD2” refers to cluster of differentiation 2, well-known in theart. The term “CD2” as provided herein includes any of the naturallyoccurring forms, homologs or variants that maintain the activity of CD2(e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to the native protein). In some embodiments, variantsor homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% aminoacid sequence identity across the whole sequence or a portion of thesequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring form. In embodiments, the CD2 proteinis the protein as identified by the Uniprot accession no. P06729 or NCBIsequence reference NP_001758.2 GI:156071472, homolog or functionalfragment thereof. CD2 is a cell adhesion molecule normally found on thesurface of T cells and natural killer (NK) cells.

The term “CD3” refers to cluster of differentiation 3, well-known in theart, and includes any one of the CD3γ, CD3δ or CD3ε chains. The term“CD3” as provided herein includes any of the naturally occurring forms,homologs or variants that maintain the activity of CD3 (e.g., within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto the native protein). In some embodiments, variants or homologs haveat least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring form. In embodiments, the CD3γ protein is theprotein as identified by the NCBI sequence reference NM_000073.2 GI:166362738, homolog or functional fragment thereof. In embodiments, theCD3δ protein is the protein as identified by the NCBI sequence referenceNM_000732.4 GI:98985799, homolog or functional fragment thereof. Inembodiments, the CD3ε protein is the protein as identified by the NCBIsequence reference NM_000733.3 GI: 166362733, homolog or functionalfragment thereof. Cd3 is a T-cell co-receptor that helps activate T-cellcytotoxicity.

The term “CD4” refers to cluster of differentiation 4, well-known in theart. The term “CD4” as provided herein includes any of the naturallyoccurring forms, homologs or variants that maintain the activity of CD4(e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to the native protein). In some embodiments, variantsor homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% aminoacid sequence identity across the whole sequence or a portion of thesequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring form. In embodiments, the CD4 proteinis the protein as identified by the UniProt accession no. 901730 or NCBIsequence reference NP_000607.1 GI: 10835167, homolog or functionalfragment thereof. CD4 is a glycoprotein found on the surface of immunecells such as T-helper cells, monocytes, macrophages and dendriticcells.

The term “CD7” refers to cluster of differentiation 7, well-known in theart. The term “CD7” as provided herein includes any of the naturallyoccurring forms, homologs or variants that maintain the activity of CD7(e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to the native protein). In some embodiments, variantsor homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% aminoacid sequence identity across the whole sequence or a portion of thesequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring form. In embodiments, the CD7 proteinis the protein as identified by the NCBI sequence reference NP_006128.1GI:5453613, homolog or functional fragment thereof. CD7 is atransmembrane protein found on thymocytes and mature T cells.

The term “CD8” refers to cluster of differentiation 8, well-known in theart, and includes any one of the CD8a or CD8b chains. The term “CD8” asprovided herein includes any of the naturally occurring forms, homologsor variants that maintain the activity of CD8 (e.g., within at least50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to thenative protein). In some embodiments, variants or homologs have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurringform. In embodiments, the CD8a protein is the protein as identified bythe NCBI sequence reference NM_001768.6 GI:225007534, homolog orfunctional fragment thereof. In embodiments, the CD8b protein is theprotein as identified by the NCBI sequence reference AAI00912.1 GI:71682667, homolog or functional fragment thereof. CD8 is a transmembraneglycoprotein co-receptor for the T-cell receptor.

The term “PD-1” refers to programmed cell death 1, well-known in theart. Programmed cell death 1 (PD-1), also called CD279, is a type Imembrane protein encoded in humans by the PDCD1 gene. It has twoligands, PD-L1 and PD-L2. The term “PD-1” as provided herein includesany of the naturally occurring forms, homologs or variants that maintainthe activity of PD-1 (e.g., within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to the native protein). In someembodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous aminoacid portion) compared to a naturally occurring form. In embodiments,the PD-1 protein is the protein as identified by UniProt accession no.Q15116 or the NCBI sequence reference NP_005009.2 GI:167857792, homologor functional fragment thereof. The PD-1 pathway is a keyimmune-inhibitory mediator of T-cell exhaustion. Blockade of thispathway can lead to T-cell activation, expansion, and enhanced effectorfunctions. As such, PD-1 negatively regulates T cell responses. PD-1 hasbeen identified as a marker of exhausted T cells in chronic diseasestates, and blockade of PD-1:PD-1L interactions has been shown topartially restore T cell function. (Sakuishi et al., JEM Vol. 207, Sep.27, 2010, pp 2187-2194). Nivolumab (BMS-936558) is an anti-PD-1 antibodythat was approved for the treatment of melanoma in Japan in July 2014.Other anti-PD-1 antibodies are described in WO 2010/077634, WO2006/121168, WO2008/156712 and WO2012/135408.

The term “CTLA4” refers to cytotoxic T-lymphocyte-associated protein 4(also called CD152), well-known in the art. It is a protein receptorfound on the surface of T cells acting as an immune checkpoint. The term“CTLA4” as provided herein includes any of the naturally occurringforms, homologs or variants that maintain the activity of CTLA4 (e.g.,within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to the native protein). In some embodiments, variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring form. In embodiments, the CTLA4 protein is theprotein as identified by UniProt accession no. P16410 or the NCBIsequence reference NP_001032720.1 GI:83700231, homolog or functionalfragment thereof.

The term “C/EBPa” or “C/EBPalpha” as provided herein includes any of theCCAAT (cytosine-cytosine-adenosine-adensoine-thymidine)/enhancer-bindingprotein alpha (C/EBPa) naturally occurring forms, homologs or variantsthat maintain the transcription factor activity of C/EBPalpha (e.g.,within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to the native protein). In some embodiments, variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring form. In embodiments, the C/EBPalpha protein is theprotein as identified by the NCBI sequence reference GI:551894998. Inembodiments, the C/EBPalpha protein is the protein as identified by theNCBI sequence reference GI:551894998, homolog or functional fragmentthereof. In embodiments, the C/EBPalpha protein is encoded by a nucleicacid sequence corresponding to Gene ID: GI:551894997.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaryotic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., Spodoptera) and human cells.

“Anti-cancer agent” is used in accordance with its plain ordinarymeaning and refers to a composition (e.g. compound, drug, antagonist,inhibitor, modulator) having antineoplastic properties or the ability toinhibit the growth or proliferation of cells. In embodiments, ananticancer agent is a chemotherapeutic. In embodiments, an anti-canceragent is an agent identified herein having utility in methods oftreating cancer. In embodiments, an anti-cancer agent is an agentapproved by the FDA or similar regulatory agency of a country other thanthe USA, for treating cancer. Examples of anti-cancer agents include,but are not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2)inhibitors (e.g. XL518, CI-1040, PD035901, selumetinib/AZD6244, GSK1120212/trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901,U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylatingagents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan,melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogenmustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil,meiphalan), ethylenimine and methylmelamines (e.g., hexamethlymelamine,thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine, lomusitne, semustine, streptozocin), triazenes(decarbazine)), anti-metabolites (e.g., 5-azathioprine, leucovorin,capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folicacid analog (e.g., methotrexate), or pyrimidine analogs (e.g.,fluorouracil, floxouridine, Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g.,vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin,paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g.,irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate,teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin,daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g.cisplatin, oxaloplatin, carboplatin), anthracenedione (e.g.,mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazinederivative (e.g., procarbazine), or adrenocortical suppressant (e.g.,mitotane, aminoglutethimide), epipodophyllotoxins (e.g., etoposide).

Further examples of anti-cancer agents include, but are not limited to,antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g.,L-asparaginase), inhibitors of mitogen activated protein kinasesignaling (e.g. U0126, PD98059, PD-184352, PD0325901, ARRY-142886,SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002), mTORinhibitors, antibodies (e.g., rituxan), 5-aza-2′-deoxycytidine,doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec®),geldanamycin, l 7-N-Allylamino-17-Demethoxygeldanamycin (17-AAG),bortezomib, trastuzumab, anastrozole; angiogenesis inhibitors;antiandrogen, antiestrogen; antisense oligonucleotides; apoptosis genemodulators; apoptosis regulators; arginine deaminase; BCR/ABLantagonists; beta lactam derivatives; bFGF inhibitor; bicalutamide;camptothecin derivatives; casein kinase inhibitors (ICOS); clomifeneanalogues; cytarabine dacliximab; dexamethasone; estrogen agonists;estrogen antagonists; etanidazole; etoposide phosphate; exemestane;fadrozole; finasteride; fludarabine; fluorodaunorunicin hydrochloride;gadolinium texaphyrin; gallium nitrate; gelatinase inhibitors;gemcitabine; glutathione inhibitors; hepsulfam; immunostimulantpeptides; insulin-like growth factor-I receptor inhibitor; interferonagonists; interferons; interleukins; letrozole; leukemia inhibitingfactor; leukocyte alpha interferon; leuprolide+estrogen+progesterone;leuprorelin; matrilysin inhibitors; matrix metalloproteinase inhibitors;MIF inhibitor; mifepristone; mismatched double stranded RNA; monoclonalantibody; mycobacterial cell wall extract; nitric oxide modulators;oxaliplatin; panomifene; pentrozole; phosphatase inhibitors; plasminogenactivator inhibitor; platinum complex; platinum compounds; prednisone;proteasome inhibitors; protein A-based immune modulator; protein kinaseC inhibitor; protein kinase C inhibitors, protein tyrosine phosphataseinhibitors; purine nucleoside phosphorylase inhibitors; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;ribozymes; signal transduction inhibitors; signal transductionmodulators; single chain antigen-binding protein; stem cell inhibitor;stem-cell division inhibitors; stromelysin inhibitors; syntheticglycosaminoglycans; tamoxifen methiodide; telomerase inhibitors; thyroidstimulating hormone; translation inhibitors; tyrosine kinase inhibitors;urokinase receptor antagonists; steroids (e.g., dexamethasone),finasteride, aromatase inhibitors, gonadotropin-releasing hormoneagonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids(e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate,megestrol acetate, medroxyprogesterone acetate), estrogens (e.g.,diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen),androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen(e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guerin(BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to 111In, 90Y, or 131I,etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin,epirubicin, topotecan, itraconazole, vindesine, cerivastatin,vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan,clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib,gefitinib, EGFR inhibitors, epidermal growth factor receptor(EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa™),erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™),panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992,CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306,ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethylerlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002,WZ3146, AG-490, XL647, PD-153035, BMS-599626), sorafenib, imatinib,sunitinib, dasatinib, or the like.

“Chemotherapeutic” or “chemotherapeutic agent” is used in accordancewith its plain ordinary meaning and refers to a chemical composition orcompound having antineoplastic properties or the ability to inhibit thegrowth or proliferation of cells.

Additionally, the nucleic acid compound described herein can beco-administered with or covalently attached to conventionalimmunotherapeutic agents including, but not limited to, immunostimulants(e.g., Bacillus Calmette-Guerin (BCG), levamisole, interleukin-2,alphainterferon, etc.), monoclonal antibodies (e.g., anti-CD20,anti-HER2, anti-CD52, anti-HLA-DR, anti-PD-1 and anti-VEGF monoclonalantibodies), immunotoxins (e.g., anti-CD33 monoclonalantibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to 111 In, 90 Y, or131I, etc.).

In a further embodiment, the nucleic acid compounds described herein canbe coadministered with conventional radiotherapeutic agents including,but not limited to, radionucleotides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr,⁸⁶Y, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁸⁶Re, ²¹¹At and ²¹²Bi, optionally conjugated to antibodies directedagainst tumor antigens.

The term “sample” includes sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histological purposes.Such samples include blood and blood fractions or products (e.g., bonemarrow, serum, plasma, platelets, red blood cells, and the like),sputum, tissue, cultured cells (e.g., primary cultures, explants, andtransformed cells), stool, urine, other biological fluids (e.g.,prostatic fluid, gastric fluid, intestinal fluid, renal fluid, lungfluid, cerebrospinal fluid, and the like), etc. A sample is typicallyobtained from a “subject” such as a eukaryotic organism, most preferablya mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; arodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; orfish. In some embodiments, the sample is obtained from a human.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Acontrol can also represent an average value gathered from a number oftests or results. One of skill in the art will recognize that controlscan be designed for assessment of any number of parameters. For example,a control can be devised to compare therapeutic benefit based onpharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of side effects). One of skill in the art will understandwhich controls are valuable in a given situation and be able to analyzedata based on comparisons to control values. Controls are also valuablefor determining the significance of data. For example, if values for agiven parameter are widely variant in controls, variation in testsamples will not be considered as significant.

“Disease” or “condition” refers to a state of being or health status ofa patient or subject capable of being treated with a compound,pharmaceutical composition, or method provided herein. In preferredembodiments, the disease is cancer (e.g. pancreatic cancer, prostatecancer, renal cancer, metastatic cancer, melanoma, castration-resistantprostate cancer, breast cancer, triple negative breast cancer,glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma(e.g., head, neck, or esophagus), colorectal cancer, leukemia, acutemyeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma), aninfectious disease (e.g., HIV infection), an inflammatory disease (e.g.,rheumatoid arthritis) or a metabolic disease (e.g., diabetes). Inembodiments, the disease is a disease related to (e.g. caused by) anaberrant activity of HSP70 (e.g. mHSP70), HSP70 (e.g. mHSP70)phosphorylation, or HSP70 (e.g. mHSP70) pathway activity, or pathwayactivated by HSP70. In some embodiments, the disease is cancer (e.g.pancreatic cancer, prostate cancer, renal cancer, metastatic cancer,melanoma, castration-resistant prostate cancer, breast cancer, triplenegative breast cancer, glioblastoma, ovarian cancer, lung cancer,squamous cell carcinoma (e.g., head, neck, or esophagus), colorectalcancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, ormultiple myeloma).

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals, including leukemia,lymphoma, carcinomas and sarcomas. Exemplary cancers that may be treatedwith a compound, pharmaceutical composition, or method provided hereininclude lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor,cervical cancer, colon cancer, esophageal cancer, gastric cancer, headand neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia,prostate cancer, breast cancer (e.g. triple negative, ER positive, ERnegative, chemotherapy resistant, herceptin resistant, HER2 positive,doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobularcarcinoma, primary, metastatic), ovarian cancer, pancreatic cancer,liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g.non-small cell lung carcinoma, squamous cell lung carcinoma,adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma,carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostatecancer, castration-resistant prostate cancer, breast cancer, triplenegative breast cancer, glioblastoma, ovarian cancer, lung cancer,squamous cell carcinoma (e.g., head, neck, or esophagus), colorectalcancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, ormultiple myeloma. Additional examples include, cancer of the thyroid,endocrine system, brain, breast, cervix, colon, head & neck, esophagus,liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary,sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma,glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primarythrombocytosis, primary macroglobulinemia, primary brain tumors, cancer,malignant pancreatic insulanoma, malignant carcinoid, urinary bladdercancer, premalignant skin lesions, testicular cancer, lymphomas, thyroidcancer, neuroblastoma, esophageal cancer, genitourinary tract cancer,malignant hypercalcemia, endometrial cancer, adrenal cortical cancer,neoplasms of the endocrine or exocrine pancreas, medullary thyroidcancer, medullary thyroid carcinoma, melanoma, colorectal cancer,papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease ofthe Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma,cancer of the pancreatic stellate cells, cancer of the hepatic stellatecells, or prostate cancer.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or a leukemic(subleukemic). Exemplary leukemias that may be treated with a compound,pharmaceutical composition, or method provided herein include, forexample, acute nonlymphocytic leukemia, chronic lymphocytic leukemia,acute granulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, or undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas that may be treated with a compound, pharmaceuticalcomposition, or method provided herein include a chondrosarcoma,fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft partsarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma,chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcomaof B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, or telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas that may betreated with a compound, pharmaceutical composition, or method providedherein include, for example, acral-lentiginous melanoma, amelanoticmelanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma,Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, nodular melanoma, subungal melanoma, or superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas that may be treated with acompound, pharmaceutical composition, or method provided herein include,for example, medullary thyroid carcinoma, familial medullary thyroidcarcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma,adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenalcortex, alveolar carcinoma, alveolar cell carcinoma, basal cellcarcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamouscell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,bronchogenic carcinoma, cerebriform carcinoma, cholangiocellularcarcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma,corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinomacutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductcarcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatinifomi carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lobularcarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinomavillosum.

As used herein, the terms “metastasis,” “metastatic,” and “metastaticcancer” can be used interchangeably and refer to the spread of aproliferative disease or disorder, e.g., cancer, from one organ oranother non-adjacent organ or body part. Cancer occurs at an originatingsite, e.g., breast, which site is referred to as a primary tumor, e.g.,primary breast cancer. Some cancer cells in the primary tumor ororiginating site acquire the ability to penetrate and infiltratesurrounding normal tissue in the local area and/or the ability topenetrate the walls of the lymphatic system or vascular systemcirculating through the system to other sites and tissues in the body. Asecond clinically detectable tumor formed from cancer cells of a primarytumor is referred to as a metastatic or secondary tumor. When cancercells metastasize, the metastatic tumor and its cells are presumed to besimilar to those of the original tumor. Thus, if lung cancermetastasizes to the breast, the secondary tumor at the site of thebreast consists of abnormal lung cells and not abnormal breast cells.The secondary tumor in the breast is referred to a metastatic lungcancer. Thus, the phrase metastatic cancer refers to a disease in whicha subject has or had a primary tumor and has one or more secondarytumors. The phrases non-metastatic cancer or subjects with cancer thatis not metastatic refers to diseases in which subjects have a primarytumor but not one or more secondary tumors. For example, metastatic lungcancer refers to a disease in a subject with or with a history of aprimary lung tumor and with one or more secondary tumors at a secondlocation or multiple locations, e.g., in the breast.

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease (e.g.,diabetes, cancer (e.g. prostate cancer, renal cancer, metastatic cancer,melanoma, castration-resistant prostate cancer, breast cancer, triplenegative breast cancer, glioblastoma, ovarian cancer, lung cancer,squamous cell carcinoma (e.g., head, neck, or esophagus), colorectalcancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, ormultiple myeloma)) means that the disease (e.g., diabetes, cancer (e.g.prostate cancer, renal cancer, metastatic cancer, melanoma,castration-resistant prostate cancer, breast cancer, triple negativebreast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cellcarcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia,acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma)or viral disease (e.g., HIV infection associated disease)) is caused by(in whole or in part), or a symptom of the disease is caused by (inwhole or in part) the substance or substance activity or function.

The term “aberrant” as used herein refers to different from normal. Whenused to describe enzymatic activity, aberrant refers to activity that isgreater or less than a normal control or the average of normalnon-diseased control samples. Aberrant activity may refer to an amountof activity that results in a disease, wherein returning the aberrantactivity to a normal or non-disease-associated amount (e.g. by using amethod as described herein), results in reduction of the disease or oneor more disease symptoms.

In some embodiments, the cancer is one in which at least one of HSP70,vimentin, HSP90, TfR or PDGFR-a expression is upregulated(overexpressed). For example HSP70 is constitutively overexpressed inpancreatic cancer cells (Hyun et al., Gut Liver. 2013 November;7(6):739-46).

Upregulation of expression comprises expression of at least one of(optionally only one of) HSP70, vimentin, HSP90, TfR or PDGFR-a at alevel that is greater than would normally be expected for a cell ortissue of a given type. Upregulation may be determined by determiningthe level of expression at least one of HSP70, vimentin, HSP90, TfR orPDGFR-a in a cell or tissue. A comparison may be made between the levelof expression in a cell or tissue sample from a subject and a referencelevel of, e.g. a value or range of values representing a normal level ofexpression for the same or corresponding cell or tissue type. In someembodiments reference levels may be determined by detecting expressionin a control sample, e.g. in corresponding cells or tissue from ahealthy subject or from healthy tissue of the same subject. In someembodiments reference levels may be obtained from a standard curve ordata set.

Levels of expression may be quantitated for absolute comparison, orrelative comparisons may be made.

In some embodiments upregulation of expression may be considered to bepresent when the level of expression in the test sample is at least 1.1times that of a reference level. More preferably, the level ofexpression may be selected from one of at least 1.2, at least 1.3, atleast 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, atleast 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, atleast 2.4 at least 2.5, at least 2.6, at least 2.7, at least 2.8, atleast 2.9, at least 3.0, at least 3.5, at least 4.0, at least 5.0, atleast 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0times that of the reference level.

Expression levels may be determined by one of a number of known in vitroassay techniques, such as PCR based assays, in situ hybridisationassays, flow cytometry assays, immunological or immunohistochemicalassays.

By way of example suitable techniques involve a method of detecting thelevel of at least one of (optionally only one of) HSP70, vimentin,HSP90, TfR or PDGFR-a in a sample by contacting the sample with an agentcapable of binding at least one of HSP70, vimentin, HSP90, TfR orPDGFR-a and detecting the formation of a complex of the agent and atleast one of HSP70, vimentin, HSP90, TfR or PDGFR-a. The agent may beany suitable binding molecule, e.g. an antibody, polypeptide, peptide,oligonucleotide, aptamer or small molecule, and may optionally belabelled to permit detection, e.g. visualisation, of the complexesformed. Suitable labels and means for their detection are well known tothose in the art and include fluorescent labels (e.g. fluorescein,rhodamine, eosine and NDB, green fluorescent protein (GFP), chelates ofrare earths such as europium (Eu), terbium (Tb) and samarium (Sm),tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone,7-amino-4-methyl coumarin, Cy3, Cy5), isotope markers, radioisotopes(e.g. ³²P, ³³P, ³⁵S), chemiluminescence labels (e.g. acridinium ester,luminol, isoluminol), enzymes (e.g. peroxidase, alkaline phosphatase,glucose oxidase, beta-galactosidase, luciferase), antibodies, ligandsand receptors. Detection techniques are well known to those of skill inthe art and can be selected to correspond with the labelling agent.Suitable techniques include PCR amplification of oligonucleotide tags,mass spectrometry, detection of fluorescence or colour, e.g. uponenzymatic conversion of a substrate by a reporter protein, or detectionof radioactivity.

Assays may be configured to quantify the amount of at least one ofHSP70, vimentin, HSP90, TfR or PDGFR-a in a sample. Quantified amountsfrom a test sample may be compared with reference values, and thecomparison used to determine whether the test sample contains an amountof at least one of HSP70, vimentin, HSP90, TfR or PDGFR-a that is higheror lower than that of the reference value to a selected degree ofstatistical significance.

Quantification of detected HSP70, vimentin, HSP90, TfR or PDGFR-a may beused to determine up- or down-regulation or amplification of genesencoding HSP70, vimentin, HSP90, TfR or PDGFR-a.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules, or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated, however, that the resulting reaction product can beproduced directly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture. Contacting may include allowing twospecies to react, interact, or physically touch, wherein the two speciesmay be a nucleic acid compound as described herein and a cell (e.g.,cancer cell).

Nucleic Acid Compounds

The aptamers provided herein, including embodiments thereof, are, interalia, capable of binding cell surface protein target molecules andinternalizing into the cell. For example, mHSP70, vimentin and HSP90 areexpressed within and present on the surface of a broad variety ofdifferent cancer cells (e.g., pancreatic cancer, liver cancer, prostatecancer). CCR5, CCR7, CD2, CD3, CD4, CD7, CD8, PD-1, CTLA4 are expressedwithin and present on the surface of a variety of immune cells,including T-cells,

Therefore, the compounds (e.g., nucleic acid compounds) provided herein,including embodiments thereof, may be used to deliver therapeutic ordiagnostic molecules into a target molecule-expressing cancer cell. Thetherapeutic or diagnostic molecule may form part of the compound (e.g.,nucleic acid compound) provided herein including embodiments thereof.

Where the therapeutic or diagnostic molecule forms part (e.g., throughcovalent attachment) of the compound (e.g., nucleic acid compound)provided herein, including embodiments thereof, the therapeutic ordiagnostic molecule is referred to as a “compound moiety” (e.g.,therapeutic moiety, imaging moiety). Alternatively, the therapeutic ordiagnostic molecule may not form part of the compound (e.g., nucleicacid compound) provided herein, including embodiments thereof, but maybe independently internalized by a target molecule-expressing cell uponbinding of a compound (e.g., nucleic acid compound) provided herein tothe target molecule on said cell. Where the therapeutic or diagnosticmolecule does not form part of the compound (e.g., nucleic acidcompound) provided herein, the molecule is referred to as a “secondcompound.” The compounds (e.g., nucleic acid compounds) provided hereinincluding embodiments thereof provide highly specific and efficientmeans for targeted cancer drug delivery and molecular imaging.

Where a nucleic acid sequence has at least 80% (80% or more) sequenceidentity to a given sequence, the nucleic acid sequence may have 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to that sequence (e.g., an aptamer sequence). In embodiments,nucleic acid sequence may have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to the entire sequence ofthat sequence or to continuous portions (e.g., a 10, 20, 30, 40, 50, 60,70, 80, 90 continuous nucleotides portion) of that aptamer sequence. Insome embodiments, the nucleic acid sequence has at least 80% (80% ormore) sequence identity to a nucleic acid that hybridizes to a givensequence.

In embodiments, the nucleic acid sequence of a mono-specific aptamer orregion of a bi-specific aptamer capable of binding one of the two targetmolecules is less than 100 (99 or less) nucleotides in length. For abi-specific aptamer, two nucleic acid sequences may be present, eachnucleic acid sequence having a length as described herein. The lengthcalculation may optionally exclude nucleotides or carbon moieties of anyspacer, linker or sticky bridge that forms part of the nucleic acidmolecule. The length calculation may also optionally exclude anycompound moiety conjugated to the nucleic acid, e.g. any nucleic acidmoiety such as siRNA, saRNA, miRNA etc.

Where the nucleic sequence is less than 100 (99 or less) nucleotides inlength the sequence is one of 99, 98, 97, 96, 95, 94, 93, 92, 91, 90,89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72,71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54,53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36,35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20nucleotides in length. In embodiments, the nucleic acid sequence is lessthan 90 nucleotides in length. In embodiments, the nucleic acid sequenceis less than 80 nucleotides in length. In embodiments, the nucleic acidsequence is less than 70 nucleotides in length. In embodiments, thenucleic acid sequence is less than 60 nucleotides in length. Inembodiments, the nucleic acid sequence is less than 50 nucleotides inlength. In embodiments, the nucleic acid sequence is less than 40nucleotides in length.

In embodiments, the nucleic acid sequence is between 20 and 99nucleotides in length. In embodiments, the nucleic acid sequence isbetween 25 and 99 nucleotides in length. In embodiments, the nucleicacid sequence is between 30 and 99 nucleotides in length. Inembodiments, the nucleic acid sequence is between 35 and 99 nucleotidesin length. In embodiments, the nucleic acid sequence is between 40 and99 nucleotides in length. In embodiments, the nucleic acid sequence isbetween 45 and 99 nucleotides in length. In embodiments, the nucleicacid sequence is between 50 and 99 nucleotides in length. Inembodiments, the nucleic acid sequence is between 55 and 99 nucleotidesin length. In embodiments, the nucleic acid sequence is between 60 and99 nucleotides in length. In embodiments, the nucleic acid sequence isbetween 65 and 99 nucleotides in length. In embodiments, the nucleicacid sequence is between 70 and 99 nucleotides in length. Inembodiments, the nucleic acid sequence is between 75 and 99 nucleotidesin length. In embodiments, the nucleic acid sequence is between 80 and99 nucleotides in length. In embodiments, the nucleic acid sequence isbetween 85 and 99 nucleotides in length.

Upon binding a target molecule on the surface of a cell, the nucleicacid compound provided herein (including embodiments thereof) may beinternalized by the cell. The term “internalized,” “internalizing,” or“internalization” as provided herein refers to a composition (e.g., acompound, a nucleic acid compound, a therapeutic agent, an imagingagent) being drawn into the cytoplasm of the cell (e.g. after beingengulfed by a cell membrane). In embodiments, the cell is a malignantcell. In embodiments, the cell is a breast cancer cell. In embodiments,the cell is a prostate cancer cell. In embodiments, the cell is a livercancer cell. In embodiments, the cell is a pancreatic cancer cell. Inembodiments, the cell is a lung cancer cell. In embodiments, the cell isa leukemia cell. In embodiments, the cell is a glioblastoma cell. Inembodiments, the cell is a colon cancer cell. In embodiments, the cellis a non-malignant cell.

In some embodiments the aptamer is a deoxyribonucleic acid. In somepreferred embodiments the aptamer may be a ribonucleic acid. The aptamermay be single stranded. Aptamers according to the present invention maycontain one or more bases that are chemically modified. In someembodiments, each base of a given type (e.g. A, T, C, G) may contain thesame chemical modification.

Aptamers according to the present invention may contain one or morenucleotides that are chemically modified at the 2′ position of ribose.In some embodiments, each ribose contains the same chemicalmodification. In some other embodiments the ribose of certainnucleotides (e.g. A, T, C, G) may be independently modified. Suchmodifications may include O-methyl modification (2′-OMe), Fluoridemodification (2′-F) or amine modification (2-NH₂).

The nucleic acid compound provided herein (including embodimentsthereof) may include a compound moiety. Where the nucleic acid compoundincludes a compound moiety, the compound moiety may be covalently (e.g.directly or through a covalently bonded intermediary) attached to thenucleic acid sequence (see, e.g., useful reactive moieties or functionalgroups used for conjugate chemistries set forth above). Thus, inembodiments, the nucleic acid compound further includes a compoundmoiety covalently attached to the nucleic acid sequence. In embodiments,the compound moiety and the nucleic acid sequence form a conjugate. Inembodiments, the compound moiety is non-covalently (e.g. through ionicbond(s), van der Waal's bond(s)/interactions, hydrogen bond(s), polarbond(s), or combinations or mixtures thereof) attached to the nucleicacid sequence.

In embodiments, the compound moiety is a therapeutic moiety or animaging moiety.

In embodiments, the therapeutic moiety is covalently attached to thenucleic acid sequence. In embodiments, the imaging moiety is covalentlyattached to the nucleic acid sequence. The term “therapeutic moiety” asprovided herein is used in accordance with its plain ordinary meaningand refers to a monovalent compound having a therapeutic benefit(prevention, eradication, amelioration of the underlying disorder beingtreated) when given to a subject in need thereof. Therapeutic moietiesas provided herein may include, without limitation, peptides, proteins,nucleic acids, nucleic acid analogs, small molecules, antibodies,enzymes, prodrugs, cytotoxic agents (e.g. toxins) including, but notlimited to ricin, doxorubicin, daunorubicin, taxol, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin D, diphteria toxin, Pseudomonasexotoxin (PE) A, PE40, abrin, and glucocorticoid. In embodiments, thetherapeutic moiety is an anti-cancer agent or chemotherapeutic agent asdescribed herein. In embodiments, the therapeutic moiety is a nucleicacid moiety, a peptide moiety or a small molecule drug moiety. Inembodiments, the therapeutic moiety is a nucleic acid moiety. Inembodiments, the therapeutic moiety is a peptide moiety. In embodiments,the therapeutic moiety is a small molecule drug moiety. In embodiments,the therapeutic moiety is a nuclease. In embodiments, the therapeuticmoiety is an immunostimulator. In embodiments, the therapeutic moiety isa toxin. In embodiments, the therapeutic moiety is a nuclease. Inembodiments, the therapeutic moiety is a zinc finger nuclease. Inembodiments, the therapeutic moiety is a transcription activator-likeeffector nuclease. In embodiments, the therapeutic moiety is Cas9. Inembodiments, the therapeutic moiety is gemcitabine or a reactivefragment thereof. “Gemcitabine” as provided herein refers to thechemical compound4-amino-1-(2-deoxy-2,2-difluoro-13-D-erythropentofuranosyl)pyrimidin-2(1H)-on. In a customary sense gemcitabine refers to CASRegistry No. 95058-81-4.

In embodiments, the therapeutic moiety is an activating nucleic acidmoiety (a monovalent compound including an activating nucleic acid) oran antisense nucleic acid moiety (a monovalent compound including anantisense nucleic acid). An activating nucleic acid refers to a nucleicacid capable of detectably increasing the expression or activity of agiven gene or protein. The activating nucleic acid can increaseexpression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore in comparison to a control in the absence of the activating nucleicacid. In certain instances, expression or activity is 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 10-fold or higher than the expression oractivity in the absence of the activating nucleic acid.

In embodiments, the therapeutic moiety is an miRNA moiety (a monovalentcompound including a miRNA), an mRNA moiety (a monovalent compoundincluding an mRNA), an siRNA moiety (a monovalent compound including ansiRNA) or an saRNA moiety (a monovalent compound including an saRNA). Inembodiments, the therapeutic moiety is a miRNA moiety. The term “miRNA”is used in accordance with its plain ordinary meaning and refers to asmall non-coding RNA molecule capable of post-transcriptionallyregulating gene expression. In one embodiment, a miRNA is a nucleic acidthat has substantial or complete identity to a target gene. Inembodiments, the miRNA inhibits gene expression by interacting with acomplementary cellular mRNA thereby interfering with the expression ofthe complementary mRNA. Typically, the miRNA is at least about 15-50nucleotides in length (e.g., each complementary sequence of the miRNA is15-50 nucleotides in length, and the miRNA is about 15-50 base pairs inlength). In other embodiments, the length is 20-30 base nucleotides,preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Inembodiments, the therapeutic moiety is a siRNA moiety or saRNA moiety asdescribed herein. In embodiments, the therapeutic moiety is ananticancer agent moiety. In embodiments, the therapeutic moiety is anmRNA moiety. In embodiments, the therapeutic moiety is a siRNA moiety.In embodiments, the therapeutic moiety is a saRNA moiety. Inembodiments, the therapeutic moiety is a cDNA moiety. In embodiments,the therapeutic moiety is a C/EBPalpha saRNA moiety. A “C/EBPalphasaRNA” as provided herein is a saRNA capable of activating theexpression of a C/EBPalpha protein. In embodiments, the therapeuticmoiety is a HSP70 siRNA moiety.

The compound moiety provided herein may be an imaging moiety. An“imaging moiety” as provided herein is a monovalent compound detectableby spectroscopic, photochemical, biochemical, immunochemical, chemical,or other physical means. In embodiments, the imaging moiety iscovalently attached to the RNA sequence.

Exemplary imaging moieties are without limitation 32P, radionuclides,positron-emitting isotopes, fluorescent dyes, fluorophores, antibodies,bioluminescent molecules, chemoluminescent molecules, photoactivemolecules, metals, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), magnetic contrast agents, quantum dots,nanoparticles, biotin, digoxigenin, haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget peptide. Any method known in the art for conjugating an antibodyto the moiety may be employed, e.g., using methods described inHermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego. Exemplary fluorophores include fluorescein, rhodamine, GFP,coumarin, FITC, Alexa fluor, Cy3, Cy5, BODIPY, and cyanine dyes.Exemplary radionuclides include Fluorine-18, Gallium-68, and Copper-64.Exemplary magnetic contrast agents include gadolinium, iron oxide andiron platinum, and manganese. In embodiments, the imaging moiety is abioluminescent molecule.

In embodiments, the imaging moiety is a photoactive molecule. Inembodiments, the imaging moiety is a metal. In embodiments, the imagingmoiety is a nanoparticle.

The compound (e.g., nucleic acid compound) provided herein may include aligand moiety. A “HSP70 ligand moiety” as used herein refers to amonovalent compound (e.g. substituent) capable of binding (interacting)to a tumor cell antigen or immune cell surface protein, as describedherein. The binding may be specific relative to relative to other cellsurface proteins. In embodiments, ligand moiety is a nucleic acidmoiety, a peptide moiety or a small molecule moiety (e.g. a smallmolecule drug moiety). In embodiments, the ligand moiety forms part ofthe RNA sequence. In embodiments, the ligand moiety comprises orconsists of the sequence of SEQ ID NO:8. In embodiments, the compoundprovided herein is bound to a cellular receptor. In embodiments, acellular receptor is one of HSP70, vimentin, HSP90, TfR or PDGFR-a orone of CCR5, CCR7, CD2, CD3, CD4, CD7, CD8, PD-1, CTLA4. “Cell surface”as provided herein refers to a protein expressed (e.g. present) on thesurface of a cell (i.e. on the cell membrane accessible to theextracellular space). In embodiments, the cellular receptor is expressed(e.g. present) on a cancer cell (i.e. on the cancer cell membraneaccessible to the extracellular space). In embodiments, the cancer cellis a pancreatic cancer cell. In embodiments, the cancer cell is aglioblastoma cell. In embodiments, the cancer cell is a liver cancercell. In embodiments, the cancer cell is a prostate cancer cell. Inembodiments, the cancer cell is a breast cancer cell. In embodiments,the cancer cell is a leukemia cell. In embodiments, the cell is a coloncancer cell.

Pharmaceutical Formulations

Pharmaceutical compositions of the compounds (e.g., nucleic acidcompounds) provided herein may include compositions having a therapeuticmoiety contained in a therapeutically effective amount, i.e., in anamount effective to achieve its intended purpose.

The pharmaceutical compositions of the compounds (e.g., nucleic acidcompounds) provided herein may include compositions having imagingmoieties contained in an effective amount, i.e., in an amount effectiveto achieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated, tested, detected, or diagnosed. When administered in methods totreat a disease, such compositions will contain an amount of activeingredient effective to achieve the desired result, e.g., modulating theactivity of a target molecule, and/or reducing, eliminating, or slowingthe progression of disease symptoms. Determination of a therapeuticallyeffective amount of a therapeutic moiety provided herein is well withinthe capabilities of those skilled in the art, especially in light of thedetailed disclosure herein. When administered in methods to diagnose ordetect a disease, such compositions will contain an amount of an imagingmoiety described herein effective to achieve the desired result, e.g.,detecting the absence or presence of a target molecule, cell, or tumorin a subject. Determination of a detectable amount of an imaging moietyprovided herein is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure herein.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated, kind of concurrent treatment, complications from thedisease being treated or other health-related problems. Othertherapeutic regimens or agents can be used in conjunction with themethods and compositions described herein including embodiments thereof.Adjustment and manipulation of established dosages (e.g., frequency andduration) are well within the ability of those skilled in the art.

For any composition (e.g., the nucleic acid compounds provided,combinations of an anticancer agent and the nucleic acid compoundprovided) described herein, the therapeutically effective amount can beinitially determined from cell culture assays. Target concentrationswill be those concentrations of active compound(s) that are capable ofachieving the methods described herein, as measured using the methodsdescribed herein or known in the art. As is well known in the art,effective amounts for use in humans can also be determined from animalmodels. For example, a dose for humans can be formulated to achieve aconcentration that has been found to be effective in animals. The dosagein humans can be adjusted by monitoring effectiveness and adjusting thedosage upwards or downwards, as described above. Adjusting the dose toachieve maximal efficacy in humans based on the methods described aboveand other methods is well within the capabilities of the ordinarilyskilled artisan.

In another aspect, a pharmaceutical formulation including the nucleicacid compound as provided herein including embodiments thereof and apharmaceutically acceptable excipient is provided. In embodiments, theribonucleic acid includes a compound moiety covalently attached to thenucleic acid sequence. As described above, the compound moiety may be atherapeutic moiety or an imaging moiety covalently attached to thenucleic acid sequence.

In another aspect, the pharmaceutical formulation includes the nucleicacid compound as provided herein including embodiments thereof and atherapeutic agent. In embodiments, the nucleic acid compound and thetherapeutic agent are not covalently attached. A therapeutic agent asprovided herein refers to a composition (e.g. compound, drug,antagonist, inhibitor, modulator) having a therapeutic effect. Inembodiments, the therapeutic agent is an anticancer agent. Inembodiments, the pharmaceutical formulation includes a pharmaceuticallyacceptable excipient.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylase or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions well known in the artand include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, oxalate and the like.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

The pharmaceutical preparation is optionally in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The unit dosage form can be of a frozen dispersion.

Methods of Delivery

Provided herein are methods of delivering compounds (e.g., nucleic acidcompounds as provided herein) to a cell through binding the compound toa cell surface target molecule (e.g. HSP70, vimentin, HSP90, TfR,PDGFR-a, CCR5, CCR7, CD2, CD3, CD4, CD7, CD8, PD-1, CTLA4) andinternalizing the compound into the cell. Thus, in one aspect, a methodof delivering a compound into a cell is provided. The method includescontacting a cell surface target molecule with a compound including anucleic acid sequence capable of binding to the target molecule. Thecompound is allowed to pass into the cell thereby delivering thecompound into the cell. The passage into the cell may be facilitated(mediated) by the cell surface target molecule.

In embodiments, the cell surface target molecule is present on a cellsurface. In embodiments, the target molecule forms part of a cellularvesicle upon passage into the cell.

In embodiments, the compound includes a therapeutic agent or an imagingagent. In embodiments, the compound is a therapeutic agent or an imagingagent. In embodiments, the therapeutic agent is an antibody, a peptide,a nucleic acid or a small molecule (e.g. a drug). In embodiments, theimaging agent is a bioluminescent molecule, a photoactive molecule, ametal or a nanoparticle. In embodiments, the compound is an antibody, apeptide, a nucleic acid or a small molecule. In embodiments, thecompound is an antibody. In embodiments, the compound is a nucleic acidcompound as provided herein including embodiments thereof. Inembodiments, the method includes detecting the nucleic acid compound inthe cell thereby detecting the cell.

As described above the nucleic acid compounds provided herein includingembodiments thereof may be used to deliver compound moieties orcompounds (e.g., therapeutic agents or imaging agents) into a cell.Where a compound moiety (e.g., therapeutic moiety or imaging moiety) isdelivered into a cell, the compound moiety may be covalently attached tothe nucleic acid compound (RNA sequence) provided herein includingembodiments thereof. Upon binding of the nucleic acid compound (RNAsequence) to a ligand on a cell, the compound moiety is internalized bythe cell while being covalently attached to the nucleic acid compound(RNA sequence). Thus, in one aspect, a method of delivering a compoundmoiety into a cell is provided. The method includes, (i) contacting acell with the nucleic acid compound as provided herein includingembodiments thereof and (ii) allowing the nucleic acid compound to bindto a ligand on the cell and pass into the cell thereby delivering thecompound moiety into the cell.

Alternatively, where a compound is delivered into a cell, the compound(e.g., a therapeutic agent or an imaging agent) may not be covalentlyattached to the nucleic acid compound (nucleic acid sequence). Uponbinding of the nucleic acid compound provided herein includingembodiments thereof to the target molecule on a cell, the nucleic acidcompound and the compound provided are internalized by the cell withoutbeing covalently attached to each other.

Thus, in another aspect, a method of delivering a compound into a cellis provided. The method includes (i) contacting a cell with a compoundand the nucleic acid compound as provided herein including embodimentsthereof and (ii) allowing the nucleic acid compound to bind to a targetmolecule on the cell and the compound to pass into the cell therebydelivering the compound into the cell. In embodiments, the compound is atherapeutic agent or imaging agent. In embodiments, the compound isnon-covalently attached to the nucleic acid compound.

Methods of Treatment

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably herein. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made. Treatment includespreventing the disease, that is, causing the clinical symptoms of thedisease not to develop by administration of a protective compositionprior to the induction of the disease; suppressing the disease, that is,causing the clinical symptoms of the disease not to develop byadministration of a protective composition after the inductive event butprior to the clinical appearance or reappearance of the disease;inhibiting the disease, that is, arresting the development of clinicalsymptoms by administration of a protective composition after theirinitial appearance; preventing re-occurring of the disease and/orrelieving the disease, that is, causing the regression of clinicalsymptoms by administration of a protective composition after theirinitial appearance. For example, certain methods herein treat cancer(e.g. prostate cancer, renal cancer, metastatic cancer, melanoma,castration-resistant prostate cancer, breast cancer, triple negativebreast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cellcarcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia,acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma).For example certain methods herein treat cancer by decreasing orreducing or preventing the occurrence, growth, metastasis, orprogression of cancer; or treat cancer by decreasing a symptom ofcancer. Symptoms of cancer (e.g. prostate cancer, renal cancer,metastatic cancer, melanoma, castration-resistant prostate cancer,breast cancer, triple negative breast cancer, glioblastoma, ovariancancer, lung cancer, squamous cell carcinoma (e.g., head, neck, oresophagus), colorectal cancer, leukemia, acute myeloid leukemia,lymphoma, B cell lymphoma, or multiple myeloma) would be known or may bedetermined by a person of ordinary skill in the art.

Where combination treatments are contemplated, it is not intended thatthe agents (i.e. nucleic acid compounds) described herein be limited bythe particular nature of the combination. For example, the agentsdescribed herein may be administered in combination as simple mixturesas well as chemical hybrids. An example of the latter is where the agentis covalently linked to a targeting carrier or to an activepharmaceutical. Covalent binding can be accomplished in many ways, suchas, though not limited to, the use of a commercially availablecross-linking agent.

An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g. achieve the effect for which it is administered, treat adisease, reduce enzyme activity, reduce one or more symptoms of adisease or condition, reduce viral replication in a cell). An example ofan “effective amount” is an amount sufficient to contribute to thetreatment, prevention, or reduction of a symptom or symptoms of adisease, which could also be referred to as a “therapeutically effectiveamount.” A “reduction” of a symptom or symptoms (and grammaticalequivalents of this phrase) means decreasing of the severity orfrequency of the symptom(s), or elimination of the symptom(s). A“prophylactically effective amount” of a drug is an amount of a drugthat, when administered to a subject, will have the intendedprophylactic effect, e.g., preventing or delaying the onset (orreoccurrence) of an injury, disease, pathology or condition, or reducingthe likelihood of the onset (or reoccurrence) of an injury, disease,pathology, or condition, or their symptoms. The full prophylactic effectdoes not necessarily occur by administration of one dose, and may occuronly after administration of a series of doses. Thus, a prophylacticallyeffective amount may be administered in one or more administrations. An“activity decreasing amount,” as used herein, refers to an amount ofantagonist required to decrease the activity of an enzyme or proteinrelative to the absence of the antagonist. A “function disruptingamount,” as used herein, refers to the amount of antagonist required todisrupt the function of an enzyme or protein relative to the absence ofthe antagonist. Guidance can be found in the literature for appropriatedosages for given classes of pharmaceutical products. For example, forthe given parameter, an effective amount will show an increase ordecrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%,90%, or at least 100%. Efficacy can also be expressed as “-fold”increase or decrease. For example, a therapeutically effective amountcan have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effectover a control. The exact amounts will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

“Patient” or “subject in need thereof” refers to a living organismsuffering from or prone to a disease or condition that can be treated byusing the methods provided herein. The term does not necessarilyindicate that the subject has been diagnosed with a particular disease,but typically refers to an individual under medical supervision.Non-limiting examples include humans, other mammals, bovines, rats,mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammaliananimals. In embodiments, a patient is human.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intrathecal, intranasalor subcutaneous administration, or the implantation of a slow-releasedevice, e.g., a mini-osmotic pump, to a subject. Administration is byany route, including parenteral and transmucosal (e.g., buccal,sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).

Parenteral administration includes, e.g., intravenous, intramuscular,intra-arteriole, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial. Other modes of delivery include, butare not limited to, the use of liposomal formulations, intravenousinfusion, transdermal patches, etc. By “co-administer” it is meant thata composition described herein is administered at the same time, justprior to, or just after the administration of one or more additionaltherapies, for example cancer therapies such as chemotherapy, hormonaltherapy, radiotherapy, or immunotherapy. The compounds of the inventioncan be administered alone or can be coadministered to the patient.Coadministration is meant to include simultaneous or sequentialadministration of the compounds individually or in combination (morethan one compound). Thus, the preparations can also be combined, whendesired, with other active substances (e.g. to reduce metabolicdegradation). The compositions of the present invention can be deliveredby transdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

In another aspect, a method of treating cancer is provided. The methodincludes administering to a subject in need thereof an effective amountof the nucleic acid compound as provided herein (including embodimentsthereof). In embodiments, the nucleic acid compound further includes ananticancer therapeutic moiety. In another aspect, a method of treatingcancer is provided. The method includes administering to a subject inneed thereof an effective amount of an anticancer agent and a nucleicacid compound as provided herein including embodiments thereof.

Methods of Detecting a Cell

The nucleic acid compositions provided herein may also be used for thedelivery of compounds and compound moieties to a cell expressing atarget molecule (e.g. HSP70, vimentin, HSP90, TfR, PDGFR-a, CCR5, CCR7,CD2, CD3, CD4, CD7, CD8, PD-1, CTLA4). As described above, the compoundsand compound moieties delivered may be imaging agents useful for celldetections. Thus, in one aspect, a method of detecting a cell isprovided. The method includes (i) contacting a cell with the nucleicacid compound as provided herein including embodiments thereof, whereinthe nucleic acid compound further includes an imaging moiety, (ii) thenucleic acid compound is allowed to bind to the target molecule on thecell and pass into the cell, (iii) the imaging moiety is detectedthereby detecting the cell.

In another aspect, a method of detecting a cell is provided. The methodincludes (i) contacting a cell with an imaging agent and the nucleicacid compound as provided herein including embodiments thereof. (ii) Thenucleic acid compound is allowed to bind to the target molecule on thecell and the imaging agent is allowed to pass into the cell. (iii) Theimaging agent is detected thereby detecting the cell.

In embodiments, the cell is a malignant cell. In embodiments, the cellis a breast cancer cell. In embodiments, the cell is a prostate cancercell. In embodiments, the cell is a liver cancer cell. In embodiments,the cell is a pancreatic cancer cell. In embodiments, the cancer cell isa glioblastoma cell. In embodiments, the cell is a colon cancer cell. Inembodiments, the cell is a non-malignant cell. In embodiments, the cellforms part of an organism. In embodiments, the organism is a mammal. Inembodiments, the cell forms part of a cell culture.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

EMBODIMENTS

Embodiments contemplated herein include embodiments P1 to P41 following.

Embodiment P1

A bi-specific aptamer capable of binding a tumor cell antigen and animmune cell surface protein.

Embodiment P2

The bi-specific aptamer of embodiment P1, wherein the tumor cell antigenis HSP70, vimentin, HSP90, TfR or PDGFR-a.

Embodiment P3

The bi-specific aptamer of embodiment P1 or P2, wherein the immune cellsurface protein is selected from the group consisting of CCR5, CCR7,CD2, CD3, CD4, CD7, CD8, PD-1, CTLA4.

Embodiment P4

A bi-specific aptamer capable of binding a cancer cell and an immunecell.

Embodiment P5

A bi-specific aptamer capable of binding a pancreatic cancer cell and animmune cell.

Embodiment P6

The bi-specific aptamer of embodiment P4 or P5, wherein the immune cellis a T-cell.

Embodiment P7

A bi-specific aptamer capable of binding HSP70 and an immune cellsurface protein.

Embodiment P8

A bi-specific aptamer capable of binding vimentin and an immune cellsurface protein.

Embodiment P9

A bi-specific aptamer capable of binding HSP90 and an immune cellsurface protein.

Embodiment P10

The bi-specific aptamer of any one of embodiments P4 to P9, wherein theimmune cell surface protein is selected from the group consisting ofCCR5, CCR7, CD2, CD3, CD4, CD7, CD8, PD-1, CTLA4.

Embodiment P11

A bi-specific aptamer capable of binding HSP70 and CCR5.

Embodiment P12

A bi-specific aptamer capable of binding vimentin and CCR5.

Embodiment P13

A bi-specific aptamer capable of binding HSP90 and CCR5.

Embodiment P14

The bi-specific aptamer according to any one of the precedingembodiments, wherein the HSP70 is mHSP70.

Embodiment P15

The bi-specific aptamer of any one of embodiments P1 to P14, wherein thebi-specific aptamer comprises the nucleic acid sequence of one of SEQ IDNOs:1 to 7.

Embodiment P16

The bi-specific aptamer of any one of embodiments P1 to P14, wherein thebi-specific aptamer comprises a nucleic acid sequence having at least80% sequence identity to one of SEQ ID NOs:1 to 7.

Embodiment P17

The bi-specific aptamer of any one of embodiments P1 to P16, wherein thebi-specific aptamer comprises the nucleic acid sequence of SEQ ID NO: 8.

Embodiment P18

The bi-specific aptamer of any one of embodiments P1 to P14, wherein thebi-specific aptamer comprises the nucleic acid sequence of one of SEQ IDNOs:28 to 30.

Embodiment P19

The bi-specific aptamer of any one of embodiments P1 to P14, wherein thebi-specific aptamer comprises a nucleic acid sequence having at least80% sequence identity to one of SEQ ID NOs:28 to 30.

Embodiment P20

The bi-specific aptamer of any one of embodiments P1 to P14, wherein thebi-specific aptamer comprises the nucleic acid sequence of one of SEQ IDNOs:31 or 32.

Embodiment P21

The bi-specific aptamer of any one of embodiments P1 to P14, wherein thebi-specific aptamer comprises a nucleic acid sequence having at least80% sequence identity to one of SEQ ID NOs:31 or 32.

Embodiment P22

The bi-specific aptamer of any one of embodiments P1 to P21, wherein thebi-specific aptamer comprises the nucleic acid sequence of one of SEQ IDNOs:9 to 16.

Embodiment P23

The bi-specific aptamer of any one of embodiments P1 to P21, wherein thebi-specific aptamer comprises a nucleic acid sequence having at least80% sequence identity to one of SEQ ID NOs:9 to 16.

Embodiment P24

A bi-specific aptamer comprising one of SEQ ID NOs:17, or 19 to 24.

Embodiment P25

A bi-specific aptamer comprising SEQ ID NO: 18.

Embodiment P26

A bi-specific aptamer comprising a complex of one of one of SEQ IDNOs:17, or 19 to 24 and SEQ ID NO:18.

Embodiment P27

The bi-specific aptamer of any one of embodiments P1 to P26 wherein oneor more bases or nucleotides are chemically modified.

Embodiment P28

The bi-specific aptamer of any one of embodiments P1 to P27 wherein oneor more nucleotides are chemically modified at the 2′ position ofribose.

Embodiment P29

A complex, optionally an in vitro complex, of a bi-specific aptameraccording to any one of embodiments P1 to P28 and a tumor cellexpressing a tumor cell antigen to which the bi-specific aptamer iscapable of binding.

Embodiment P30

A complex, optionally an in vitro complex, of a bi-specific aptameraccording to any one of embodiments P1 to P28 and an immune cellexpressing an immune cell surface protein to which the bi-specificaptamer is capable of binding.

Embodiment P31

A complex, optionally an in vitro complex, of a bi-specific aptameraccording to any one of embodiments P1 to P28, a tumor cell expressing atumor cell antigen to which the bi-specific aptamer is capable ofbinding and an immune cell expressing an immune cell surface protein towhich the bi-specific aptamer is capable of binding.

Embodiment P32

The complex of embodiment P30 or P31, wherein the immune cell is aT-cell.

Embodiment P33

A pharmaceutical composition comprising a bi-specific aptamer accordingto any one of embodiments P1 to P28 and a pharmaceutically acceptablecarrier, diluent or excipient.

Embodiment P34

A bi-specific aptamer according to any one of embodiments P1 to P28 foruse in a method of medical treatment.

Embodiment P35

A bi-specific aptamer according to any one of embodiments P1 to P28 foruse in a method of treatment of cancer.

Embodiment P36

The bi-specific aptamer for use in a method of treatment of canceraccording to embodiment P35, wherein the cancer is a pancreatic cancer.

Embodiment P37

The bi-specific aptamer for use in a method of treatment of canceraccording to embodiment P35 or P36, wherein the cancer overexpresses atleast one of HSP70, vimentin, HSP90, Tfr or PDGFR-a.

Embodiment P38

A method of treatment of cancer in a subject, the method comprisingadministering a therapeutically effective amount of a bi-specificaptamer according to any one of embodiments P1 to P28 to a subject inneed of treatment.

Embodiment P39

The method of embodiment P38, wherein the cancer is a pancreatic cancer.

Embodiment P40

The method of embodiment P38 or P39, wherein the cancer overexpresses atleast one of HSP70, vimentin, HSP90, Tfr or PDGFR-a.

Embodiment P41

A method of selecting a subject for treatment of cancer with atherapeutically effective amount of a bi-specific aptamer according toany one of embodiments P1 to P28, the method comprising determining, invitro, whether cells of a cancer in the subject overexpress at least oneof HSP70, vimentin, HSP90, TfR or PDGFR-a.

EXAMPLES Example 1: Bi-Specific RNA Aptamers for Targeting Cancer

We investigated the construction of cancer specific bi-specific RNAaptamers to recruit endogenous T cells. The bi-specific RNA aptamersformed a bridge between cancer cell targets and T cells, forming animmunological ‘cytolytic synapse’ (FIG. 16), leading to rapid lysis ofcancer cells.

1. Sequences

tP19 (truncated P19; pancreatic cancer aptamer) conjugated to stickysequence via polycarbon linker:

[SEQ ID NO: 17] fCfUfCAAfUGGfCGAAfUGfCfCfCGfCfCfUAAfUAGGGooooooomAmGfUfUfUfUfUfUmAfCmAfUfUfUfUmG

CCR5 aptamer (G3; T cell surface markers):

[SEQ ID NO: 18] GGGAGGAfCGAfUGfCGGGfCfCfUfUfCGfUfUfUGfUfUfUfCGfUfCfCAfCAGAfCGAfCfUfCGfCfCfCGAooooofCmAmAmAmAfUmGfUmA mAmAmAmAmAfCfU

Bold: sticky sequence. fU and fC: 2′F modified pyrimidines. mA and mG:2′O methylated purines, o: C3 carbon linker.

tP19 (SEQ ID NO:1) is an mHSP70 binding aptamer capable of internalisingupon binding of mHSP70 at the cell surface, and described inWO2013/154735. tP19 is a truncated form of aptamer P19 which also bindsmHSP70, also described in WO2013/154735. WO2013/154735 is specificallyincorporated herein by reference in its entirety. tP19 is also describedin U.S. provisional patent application No. 62/141,156, specificallyincorporated herein by reference in its entirety.

The CCR5 binding aptamer G3 (SEQ ID NO:9) is described in Zhou et al.,2015, Chemistry & Biology 22, 379-390 Mar. 19, 2015 and in co-pendingU.S. patent application Ser. No. 14/801,710, specifically incorporatedherein by reference in its entirety.

2. Formation of Bi-Specific RNA Aptamers Capable of Binding mHSP70 andCCR5.

tP19 was folded in binding buffer (phosphate-buffered saline solution[DPBS] without Ca²⁺ and Mg²⁺, 5 mM MgCl₂) at 95° C. for 5 mins andslowly cooled down. CCR5 aptamer was folded in binding buffer (DPBS withMgCl₂ and CaCl₂) at 65° C. for 5 mins and slowly cooled down. The sameconcentration of tP19 and CCR5 aptamers were mixed and incubated at 37°C. for 20 mins to make the conjugate using ‘sticky sequence’ technology(FIG. 4; M: Marker, 1:tP19 aptamer, 2: CCR5 aptamer, 3: tP19-CCR5bi-specific RNA aptamers). The ‘sticky sequence’ technology successfullyconjugated tP19 and CCR5 aptamers for bi-specific RNA aptamers.

3. Target Binding Assay

To test the binding of bi-specific RNA aptamers (tP19-CCR5 aptamer) ontarget cells which is PANC-1, live cell imaging was performed. tP19 waslabeled with Cy3 and CCR5 aptamer was labeled with FAM. 500 nM ofbi-specific RNA aptamers was incubated in PANC-1 for 4 hours and tookimages by confocal microscopy (Red: Cy3, Green: FAM, Blue: Hoechest fornuclear staining). The bi-specific RNA aptamers stayed on the surface ofPANC-1, not internalized.

4. Cytotoxic T Lymphocyte Assay (CTL) of Bi-Specific RNA Aptamers(tP19-CCR5).

To test tumor cell lysis by bi-specific RNA aptamers, health human Tcells were isolated using CD8 positive selection kits (Stem cell,#18053). After isolation, CD8 T cells enrichment was confirmed with flowcytometry.

Target cells, PANC-1 were labeled with Calcein-AM which is greenfluorescence dye in living cells. Effector cells were isolated CD8 Tcells and target cells were PANC-1. 500 nM of bi-specific RNA aptamerswere incubated with the cells in media without Phenol Red and FBS for 8hours at E:T ratio=20:1. Four different experimental groups were setupto test whether the isolated CD8 T cells attack foreign cells. tP19;tP19 aptamer in CD8 Tcells: PANC-1(E:T ratio=20:1). CCR5; CCR5 aptamerin CD8 Tcells: PANC-1(E:T ratio=20:1). tP19-CCR5; bi-specific tP19-CCR5aptamer in CD8 Tcells: PANC-1(E:T ratio=20:1). CC; without aptamers inCD8 T cells: PANC-1(E:T ratio=20:1).

The released fluorescence was measured by fluorescence plate reader.

Specific lysis was calculated by the following formulation.Specific lysis (%)=(experimental release−spontaneous releasecontrol/maximum release control−spontaneous release control)×100.

The bi-specific RNA aptamers induced PANC-1 lysis up to 80% (FIG. 9).

Real-time video microscopy showed that the bi-specific aptamer wascapable of attracting T-cells to PANC-1 cells creating a complex ofT-cell, bi-specific aptamer and PANC-1 cells that led to rapid andefficient lysis of PANC-1 cells (e.g. see FIGS. 7 and 8).

Example 2: mHSP70/CCR5 Bi-Specific Aptamer

P19 (SEQ ID NO:2) conjugated to sticky sequence via polycarbon linker:

[SEQ ID NO: 19] GGGAGAfCAAGAAfUAAAfCGfCfUfCAAfUGGfCGAAfUGfCfCfCGfCfCfUAAfUfAGGGfCGfUfUAfUGAfCfUfUGfUfUGAGfUfUfCGAfCAGGAGGfCfUfCAfCAAfCAGGfCooooooomAmGfUfUfUfUfUfUmAfC mAfUfUfUfUmG

P1 (SEQ ID NO:4) conjugated to sticky sequence via polycarbon linker:

[SEQ ID NO: 20] GGGAGAfCAAGAAfUAAAfCGfCfUfCAAfUGfCGfCfUGAAfUGfCfCfCAGfCfCGfUGAAAGfCGfUfCGAfUfUfUfCfCAfUfCfCfUfUfCGAfCAGGAGGfCfUfCAfCAAfCAGGfCooooooomAmGfUfUfUfUfUfUmA fCmAfUfUfUfUmG

CCR5 aptamer (G3):

[SEQ ID NO: 18] GGGAGGAfCGAfUGfCGGGfCfCfUfUfCGfUfUfUGfUfUfUfCGfUfCfCAfCAGAfCGAfCfUfCGfCfCfCGAooooofCmAmAmAmAfUmGfUmA mAmAmAmAmAfCfU

Bold: sticky sequence. fU and fC: 2′F modified pyrimidines. mA and mG:2′O methylated purines, o: C3 carbon linker.

P19 (SEQ ID NO:2) and P1 (SEQ ID NO:4) are mHSP70 binding aptamers, eachcapable of internalising upon binding of mHSP70 at the cell surface.They are described in WO2013/154735.

The CCR5 binding aptamer G3 (SEQ ID NO:9) is described in Zhou et al.,2015, Chemistry & Biology 22, 379-390 Mar. 19, 2015 and in co-pendingU.S. patent application Ser. No. 14/801,710.

Formation of bi-specific aptamers capable of binding mHSP70 and CCR5:P19 or P1 conjugated to the sticky sequence via a polycarbon linker isfolded in binding buffer (phosphate-buffered saline solution [DPBS]without Ca²⁺ and Mg²⁺, 5 mM MgCl₂) at 95° C. for 5 mins and slowlycooled down. CCR5 aptamer conjugated to the sticky sequence via apolycarbon linker is folded in binding buffer (DPBS with MgCl₂ andCaCl₂) at 65° C. for 5 mins and slowly cooled down. The sameconcentration of P19 or P1 and CCR5 aptamers is mixed and incubated at37° C. for 20 mins to make the bi-specific aptamer using ‘stickysequence’ technology.

Example 3: Mass-Spectrometry Based Identification of Aptamer BindingCell-Surface Proteins

One key challenge in cancer biomarker discovery is the identification oftargets that are intracellular in normal cells but are exposed on thesurface of tumor cells. Targets with these characteristics can increasethe therapeutic specificity and affinity of candidate biomarkers forcancer cells, leading to the development of anti-cancer therapeutics,diagnosis and theragnostics. Herein, we describe methods for identifyingtumor-associated proteins on living cells as cancer biomarkers, usingblind Systematic Evolution of Ligands by EXponential enrichment (SELEX)and tandem mass spectrometry. As proof of principle, a proteomicplatform to select mislocated targets specifically expressed onpancreatic cancer plasma membrane have been developed using RNAaptamers. Vimentin was identified as binding targets. This techniquecould be applied to a variety of cancer types.

1. Introduction

Aptamers, which are small structured single-stranded RNAs, are powerfuland emerging molecular tools for identifying biomarkers in cancer, asthey can be selected to recognize a wide variety of targets includingproteins, cultured cells, and whole organisms [1-6]. Aptamers aregenerated by Systematic Evolution of Ligands by EXponential enrichment(SELEX) and hold their three-dimensional structures by well-definedcomplementary nucleic acid sequences [7, 8]. As aptamers adopt complexstructures to bind targets with high affinity and specificity, theyoffer significant advantages over antibodies: better structuralstability, low toxicity, low immunogenicity and greater safety [7, 8].Moreover, the affinity and specificity of aptamers is comparable to, oreven more greater than, the affinity and specificity of antibodies [9].Another advantage over antibodies is that aptamers can be fine-tuned andreduced in size after selection through chemical synthesis.

Mass spectrometry based proteomics allows profiling of proteinidentification, providing an important source for the development ofnovel therapeutics and diagnostic targets [10]. The strength of tandemmass spectrometry (MS/MS) is that it provides extensive sequenceinformation over the whole length of a protein chain in a single seriesof experiments that involves minimal effort directed toward theseparation and purification of oligopeptide fragments [10].

The proteins which are selectively expressed on the plasma membrane ofcancer cells can be used to further our understanding of tumordevelopment. Several plasma membrane-associated proteins expressedspecifically on tumor cells have been described, including thefollowing: Heat-shock protein 70 (HSP70) [11, 12], heat-shock protein 90(HSP90) [13, 14], glucose-regulated protein 78 (GRP78) [15, 16],vimentin [17], nucleolin [18, 19], and feto-acinar pancreatic protein(FAPP) [20]. Since Berezovski et al. initiated aptamer-facilitatedbiomarker discovery (AptaBiD) [21], the identification and validation ofbiomarkers for therapeutics and the diagnosis of cancer becomesubstantially increased. This development has opened the door for drugdiscovery. For the cancer-biomarker discovery by aptamers, cell-SELEXprocedure was employed to identify new cancer surface proteins. Severalplasma membrane-associated proteins expressed specifically on tumorcells have been identified by aptamers, including the following:Alkaline phosphatase placental-like 2 (ALPPL-2) [22], siglec-5 [23],stress-induced phosphoprotein 1 (STIP1) [24], protein tyrosine kinase 7(PTK7) [25]. In pancreatic cancer, alkaline phosphatase placental-like 2(ALPPL-2) and cyclophilin B have also been reported to be novelcandidate biomarkers that are retrieved by RNA aptamers [22, 26].

Herein, we present a detailed methodology to identify putativebiomarkers on the tumor cell plasma membrane using RNA aptamers in ablind SELEX approach that involves tandem MS/MS.

2. Methods and Results

2.1. Chemicals and Materials

Ultra-purified (Milli-Q) water, Acetonitrile (CH₃CN, Burdick and JacksonHPLC grade), Ammonium bicarbonate (NH₄HCO₃, Sigma), Dithiothreitol (DTT,Sigma), lodoacetamide (IAA, Sigma), Trypsin (Promega modified trypsin,sequencing grade), Trifluroacetic acid (TFA, Sigma).

2.2. Naïve Whole-Cell SELEX (Blind SELEX)

The following cell lines were purchased from the American Type CultureCollection (ATCC) for use as targets for SELEX and internalizationassay; PANC-1 (CRL-1469), CFPAC-1 (CRL-1918), MIA PaCa-2 (CRL-1420),BxPC-3 (CRL-1687) and AsPC-1 (CRL-1682). Huh-7 cells were purchased fromJapanase Collection of Research Bioresources (JCRB). The cells werecultured according to the cell bank's instructions.

The SELEX cycle was performed basically as described by Tuerk and Gold[8]. In vitro selection was carried out essentially as described [27],with a few modifications for this study. The human pancreaticadenocarcinoma, PANC-1, cells were used as target cells for the aptamerselection. To remove background/irrelevant binding, the hepatocellularcarcinoma cell line Huh7 was used for the counter-selection step. Alibrary of 2′F RNAs was used to increase nuclease resistance and enhanceaptamer folding. To isolate 2′F RNA aptamers binding to intact cells, alibrary of approximately 4⁴⁰ different 2′F RNA molecules, containing a40-nt-long random sequence flanked by defined sequences, was screened bySELEX. For the first round, 6 nmols of the RNA library was incubated for30 minutes at 37° C. with negative (Huh7) cells in 1 ml binding buffer(phosphate-buffered saline solution [PBS] without Ca²⁺ and Mg²⁺, 5 mMMgCl₂, 0.01% BSA, yeast tRNA (100 μg/ml). The supernatant was recoveredand incubated on the target cells for 1 hour at 37° C. RNAs that boundto target cells were recovered, amplified by RT-PCR and in vitrotranscription, and used in the following selection rounds. In subsequentrounds, the RNA concentration was reduced by 10 fold and the incubationtime was reduced to create more stringent conditions. The enriched poolswere cloned after 14 cycles of selection.

A highly enriched aptamer, P15, was selected: GGGAGACAAGAATAAACGCTCAAAGTTGCGGCCCAACCGTTTAATTCAGAATAGTGTGATGCCTTCGACAGGAGGCTCA CAACAGGC (SEQID NO:36). Minimum energy structural analyses of the selected aptamerswere carried out using the NUPACK software (at website www.nupack.org)(FIG. 17A). As depicted, the calculated secondary structures of the RNAaptamers contained several stem-loop regions.

2.3. Flow Cytometry-Based Binding Assays

Aptamer binding was also assessed by flow cytometry. For the assay, thePANC-1 cells and Huh7 cells were detached using a non-enzymatic celldissociation solution, washed with PBS and suspended in binding buffer.Next, Cy3-labeled aptamers at 200 nM were added and incubated with 2×10⁵cells for 30 minutes at 37° C. Cells were washed with binding buffer andimmediately analyzed by Fortessa flow cytometry (BD). Eachflow-cytometry assay was performed in triplicate. The data were analyzedwith FlowJo software. The flow cytometry analyses of P15 confirmedenriched cell surface binding to PANC-1 cells, compared to the initialnon-selected RNA library. PANC-1 cells treated with Cy3-labelled P15aptamers demonstrated significantly higher levels of positively stainedcells (P<0.01) (FIGS. 17B-17C). The binding affinity of P15 to PANC-1cells was determined to be 16.05 nM. To verify the specificity of P15 topancreatic cancer cells, a panel of four different pancreatic cancercell lines (AsPC-1, CFPAC, MIA PaCa-2 and BxPC-3) was treated withCy3-labelled P15 aptamer. Interestingly, punctate cytoplasmic stainingwas observed in pancreatic cancer cell lines, but no staining wasobserved in negative control cells (Huh7) (FIG. 18).

To determine the apparent dissociation constant (K_(D)) of aptamers toPANC-1 cells, the mean fluorescence intensity (MFI) was calculated foreach concentration and for the unselected library controls. The valuesfor the controls were considered to be background fluorescence and weresubtracted from the values for the aptamers, as previously described bySefah et al. [28] The dissociation constants were calculated using aone-site binding model. The non-linear curve regression was performedusing Graph Pad Prism (GraphPad Software, La Jolla, Calif., USA). Thebinding affinities of P15 aptamers were measured to 16.05 nM (FIG. 17D).

2.4. Live-Cell Confocal Imaging.

For the aptamer internalization studies, 1×10⁵ cells were seeded in 35mm glass-bottom dishes (MatTek, Ashland, Mass., USA) and grown in mediumfor 24 hrs. The RNAs were labeled with Cy3 using the Cy3 Silencer siRNAlabeling kit (Ambion, Tex., USA.). Cy3-labeled RNAs were added to thecells at 100 nM and incubated for 1 hour. The images were taken using aZeiss LSM 510 Meta Inverted 2 photon confocal microscope system using aC-Apo 40×/1.2NA water immersion objective.

Cy3-labelled P15 aptamers got internalized to the target cells (PANC-1),but no staining was observed in negative cells (Huh7). To verify thespecificity of the aptamers to pancreatic cancer cells, a panel of fourdifferent pancreatic cancer cell lines AsPC-1, CFPAC, MIA PaCa-2 andBxPC-3 were treated with Cy3-labelled P15 aptamers. Punctate cytoplasmicstaining of Cy3-labeled aptamers was observed. The pattern ofcytoplasmic staining is suggestive of endocytic internalization of theaptamers.

2.5. Affinity Purification of Target Membrane Proteins Using RNAAptamer.

As the aptamer were internalized in cells, the cell membrane proteinswere isolated from the cells to identify the target epitope of theaptamer. Biotinylated aptamers, together with the associated proteincomplexes, were immobilized using a pull-down process. The selected RNAaptamers and irrelevant RNAs were labeled with biotin at their 3′ ends.The target membrane proteins were isolated using procedures described byDaniels et al. [4]. Briefly, A monolayer of cells (plated at 1×10⁶ cellsper 100 mm dish) was washed three times at 4° C. with PBS containing 1mM CaCl₂ (pH 7.4). Three ml of hypotonic buffer (10 mM Tris-HCl [pH 7.5]containing 5 mM KCl and 0.5 mM MgCl₂ with protease inhibitors) was addedto the cell monolayer for 30 minutes 4° C. before the cells wereharvested by scraping. The stripped cells were homogenized on ice in asucrose solution (0.25 M). Whole cells and nuclei were removed bycentrifugation (1,000×g for 10 minutes at 4° C.). The supernatant wasrecentrifuged to pellet the membrane component (105,000×g for 10 minutesat 4° C.). The pellet was solubilized at 4° C. with rotation inextraction buffer (10 mM Tris-HCl [pH 7.5] containing 200 mM NaCl, 0.1%[v/v] Triton X-100, 1 mM MgCl₂, 1 mM CaCl₂ and protease inhibitors). Theextracted cell-surface membrane was incubated at 4° C. for 2 hours withbiotinylated aptamers (1 nmol), irrelevant RNAs (1 nmol) andstreptavidin beads in affinity purification buffer (extraction buffercontaining 0.5 μg/μl BSA and 4% [v/v] glycerol). The beads were washedin extraction buffer for 30 minutes at 4° C. before the bound proteinwas eluted using elution buffer (10 mM Tris-HCl [pH 7.5] containing 1 MNaCl, 5 mM EDTA and 0.1% [v/v] Triton X-100).

The retrieved proteins were separated by SDS-PAGE followed by Coomassiestaining to visualize the resolved protein bands (FIG. 19A). The highestmatching peak retrieved from P15-treated cells matched a known peak forvimentin by tandem mass spectrometry (MASS-SPEC) spectrum (FIG. 19B). Toconfirm the MASS-SPEC results, a competition assay with vimentinantibodies was performed by live cell confocal and flow cytometry.Fluorescence intensity was measured by confocal microscopy. Theantibodies to vimentin significantly reduced the binding of P15 totarget cells, P<0.05 (FIG. 19C). These results strongly suggest the P15bound to plasma membrane expressing vimentin on cancer cells.

2.6. Protein Digestion.

In-gel or in-solution digestion was used for protein purification andanalyzed by mass spectrometry for peptide fingerprinting. Afterpolyacrylamide gel electrophoresis (SDS-PAGE), the aptamer-retrievedprotein bands were excised and in-gel digested with optimization [29].Briefly, the gel band was cut into small pieces and destained withdestaining solution (200 mM NH₄HCO₃ in 50% acetonitrile) for 20 minutesat 37° C. To complete the destaining, the destaining step was repeatedafter discarding the supernatant. The gel samples were dried in a vacuumconcentrator (SpeedVac). The reducing buffer (10 mM DTT in 100 mMNH₄HCO₃, prepared immediately before use) was added to the dried gelsamples and incubated for one hour at 56° C. After removal of the excessreducing buffer, the same volume of alkylating solution (100 mM IAA,prepared immediately before use) was added for 30 minutes at roomtemperature in the dark. After removal of the supernatant, the gelpieces were washed twice with 200 mM NH₄HCO₃. To dehydrate the sample,acetonitrile was added for 10 minutes at room temperature. When the gelpieces turned white, the acetonitrile was removed and the gel pieceswere re-swelled with 200 mM NH₄HCO₃ for 10 minutes at room temperature.After removal of the supernatant, the dehydration with acetonitrile wasrepeated. The gel pieces were then dried in the vacuum concentrator. Thedigestion buffer (50 ng/μl trypsin in 1 mM HCl/100 mM NH₄HCO₃, preparedimmediately before use) was added to the dried gel pieces for 10 minutesto re-swell the gel pieces. After removing the excess trypsin solutionfrom the samples, 200 mM NH₄HCO₃ was added to cover the gel pieces andthe samples were incubated overnight at 37° C. The samples were thenquenched with 1/10 volume of quenching buffer (1:9 TFA:Milli-Q water).The supernatant was saved for the next step. The extraction buffer (0.1%TFA in 60% acetonitrile) was added to cover the gel pieces and incubatedfor 40 minutes at 37° C. The supernatant containing the extractionbuffer was then extracted and combined with the quenched solutionsupernatant from the previous step. The volume of the combinedsupernatant was reduced to 20 μl by evaporation.

2.7. Liquid Chromatography Tandem MS/MS (LC-MS/MS) Q-TOF.

An Agilent 6520 Q-TOF mass spectrometer equipped with a Chip Cube sourcewas used for LC/MS/MS analyses. A C18 chip with a 43 mm analyticalcolumn and a 40 nl trapping column (ProtID-Chip-43, Agilent G4240-62005)was used. Digested samples (10-15 μl) were loaded onto the column at 6μl/minute in 99% Buffer A (0.1% Formic Acid in water)/1% Buffer B (0.1%Formic Acid in Acetonitrile) with an extra 8 μl wash volume. Thegradient was from 3% to 35% Buffer B over 8 minutes and then 35% to 90%Buffer B over 1 minute. The total run time, including injection, was 15minutes. The voltage was adjusted to the 1850V to 2000V range. The X!Tandem search engine (http://www.thegpm.org/TANDEM/index.html) was usedto search the peptide MS/MS spectrum. The dataset was then processedusing the Scaffold program (http://www.proteomesoftware.com) tovisualize the results. SWISS Prot or NCBI were used to obtain thedetailed protein annotation. The highest matching peak detected fromP15-treated cells matched a known peak for vimentin (FIG. 19B).

2.8. Competition Assays for Validation of Target.

For the aptamer-antibody competition assay, Cy3-labeled P15 aptamer wasused to compete with vimentin antibodies (Sigma, V6630). The cells(1×10⁵) were seeded in 35 mm glass-bottom dishes (MatTek, Ashland,Mass., USA) and grown in medium for 24 hours. Cells were preincubatedwith vimentin antibodies, at 1 uM, for 20 minutes before Cy3-labeledP15, at 200 nM, was added. The cells were incubated for 2 hours at 37°C. The images were taken using a Zeiss LSM 510 Meta Inverted 2-photonconfocal microscope system using a C-Apo 40×/1.2NA water immersionobjective. The arbitrary fluorescence intensity was quantified in thepresence of competitors using confocal microscopy and analyzedstatistically. Student's t test was used for statistical significanceanalysis (P<0.05) (FIG. 19C).

For competition assay by flow cytometry, PANC-1 cells were detachedusing Accutase (Sigma-Aldrich), washed with DPBS, and suspended inbinding buffer (phosphate-buffered saline solution [DPBS without Ca²⁺and Mg²⁺, Corning, Tewksbury Mass.], 5 mM MgCl₂). Next, PANC-1 cellswere pre-incubated with vimentin antibodies, at 1 μM, for 20 minutes onice. After pre-incubation with vimentin antibodies, Cy3-labeled aptamersat 200 nM of final concentration were added and incubated with 2×10⁵cells for 30 minutes ice. Cells were washed with DPBS three times andimmediately analyzed by Fortessa flow cytometry (BD Biosciences, SanJose, Calif.). 4′6′-diamidino-2-phenylindole (DAPI) (1 μg/ml) was usedto identify and exclude dead cells. Data were analyzed with FlowJosoftware (FlowJo, Ashland, Oreg.).

3. Discussion.

We have presented protein biomarker discovery platforms that use RNAaptamers. The current approach, which included aptamer selection againstcells, cell membrane protein extraction, and tandem MS/MS, has allowedfor the identification of novel proteins translocated to the plasmamembrane. Untargeted SELEX, called “blind SELEX”, allows for thegeneration of highly enriched RNA aptamers against target cells such asprimary cells, normal cells, cancer cells, and tissue-specific cells.This strategy also allows us to distinguish different cancer cell types.The enriched RNA aptamers specifically bind to the cell membraneproteins with high affinity. The cell membrane is retrieved withaffinity purification using the RNA aptamer. The proteins are thenidentified by tandem MS/MS. Because tandem MS/MS does not require thesamples be purified to a high degree of homogeneity [10] and works wellas long as the target protein is a major component of the mixture [30],it the best choice in protein biomarker discovery. When the candidatesof target proteins are identified, the interactions of RNA aptamers withproteins can be validated by surface plasmon resonance (SPR), gel shiftassays or enzyme-linked immunoassays (ELISA).

The list of currently identified cancer cell membrane-associatedproteins includes the following: HSP70, HSP90, GRP78, vimentin,nucleolin, and FAPP. HSP70 is a chaperone with ATPase activity that isfound in the intracellular compartment in normal cells [31], but istranslocated to the plasma membrane of tumor cells [11, 12]. HSP90 is amolecular chaperone protein that assists proteins in proper folding;however, it is not only localized intracellularly but also expressed onthe plasma membrane of tumor cells [13, 14]. GRP78 is an endoplasmicreticulum (ER) protein involved in protein folding that is up-regulatedand localized on the tumor cell surface [15, 16]. Nucleolin is anucleolar protein involved in the regulation of proliferation,cytokinesis, and replication [18, 19]. Nucleolin is also expressed onthe surface of tumor-related blood vessels, but not on mature vessels orcapillaries [18]. FAPP has been identified as anothermembrane-associated protein in pancreatic cancers [20, 32]. Vimentinbelongs to the group of intermediate filament proteins, which form thecytoskeleton and are associated with the nucleus, mitochondria, and ER[33]. However, vimentin expression is also correlated with theepithelial mesenchymal transition (EMT) during tumor progression [34].Vimentin was also identified in pancreatic cancer. Even though themolecular mechanisms of the presentation of these proteins on the cancersurface still need to be resolved, it might be of interest to find novelmembrane-associated proteins for targeting molecules using RNA aptamers.The approach described herein allows for the identification of novelprotein biomarkers using RNA aptamers. A key advantage of employing RNAaptamers for biomarker discovery over antibodies is their specificitytowards the target proteins, which minimizes off-target binding.

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Example 4: Vimentin/CCR5 Bi-Specific Aptamer

P15 (SEQ ID NO:3) conjugated to sticky sequence via polycarbon linker:

[SEQ ID NO: 21] GGGAGAfCAAGAAfUAAAfCGfCfUfCAAAGfUfUGfCGGfCfCfCAAfCfCGfUfUfUAAfUfUfCAGAAfUAGfUGfUGAfUGfCfCfUfUfCGAfCAGGAGGfCfUfCAfCAAfCAGGfCooooooomAmGfUfUfUfUfUfUmAfC mAfUfUfUfUmG

CCR5 aptamer (G3):

[SEQ ID NO: 18] GGGAGGAfCGAfUGfCGGGfCfCfUfUfCGfUfUfUGfUfUfUfCGfUfCfCAfCAGAfCGAfCfUfCGfCfCfCGAooooofCmAmAmAmAfUmGfUmA mAmAmAmAmAfCfU

Bold: sticky sequence. fU and fC: 2′F modified pyrimidines. mA and mG:2′O methylated purines, o: C3 carbon linker.

P15 is a vimentin binding aptamer capable of internalising upon bindingof vimentin at the cell surface, see Example 3.

The CCR5 binding aptamer G3 (SEQ ID NO:9) is described in Zhou et al.,2015, Chemistry & Biology 22, 379-390 Mar. 19, 2015 and in co-pendingU.S. patent application Ser. No. 14/801,710.

Formation of bi-specific aptamers capable of binding vimentin and CCR5:P15 conjugated to the sticky sequence via a polycarbon linker is foldedin binding buffer (phosphate-buffered saline solution [DPBS] withoutCa²⁺ and Mg²⁺, 5 mM MgCl₂) at 95° C. for 5 mins and slowly cooled down.CCR5 aptamer conjugated to the sticky sequence via a polycarbon linkeris folded in binding buffer (DPBS with MgCl₂ and CaCl₂) at 65° C. for 5mins and slowly cooled down. The same concentration of P15 and CCR5aptamers is mixed and incubated at 37° C. for 20 mins to make thebi-specific aptamer using ‘sticky sequence’ technology.

Example 5: HSP90/CCR5 Bi-Specific Aptamer

P11 (SEQ ID NO:5) conjugated to sticky sequence via polycarbon linker:

[SEQ ID NO: 22] GGGAGAfCAAGAAfUAAAfCGfCfUfCAAAfUGAfUfUGfCfCfCAfUfUfCGGfUfUAfUGfCfUfUGfCGfCfUfUfCfCfUAAAGAGfCfUfUfCfGAfCAGGAGGfCfUfCAfCAAfCAGGfCooooooomAmGfUfUfUfUfUfU mAfCmAfUfUfUfUmG

P7 (SEQ ID NO:6) conjugated to sticky sequence via polycarbon linker:

[SEQ ID NO: 23] GGGAGAfCAAGAAfUAAAfCGfCfUfCAAGGfCfCAfUGfUfUGAAfUGfCfCfCAAfCfUAAGfCfUfUfUGAGfCfUfUfUGGAGfCfUfUfCGAfCAGGAGGfCfUfCAfCAAfCAGGfCooooooomAmGfUfUfUfUfUfUmAfC mAfUfUfUfUmG

P6 (SEQ ID NO:7) conjugated to sticky sequence via polycarbon linker:

[SEQ ID NO: 24] GGGAGAfCAAGAAfUAAAfCGfCfUfCAAfCAAfUGGfAGfCGfUfUAAAfCGfUGAGfCfCAfUfUfCGAfCAGGAGGfCfUfCAfCAAfCAGGfCooooooomAmGfUfUfUfUfUfUmAfCmAfUfUfUfUmG

CCR5 aptamer (G3):

[SEQ ID NO: 18] GGGAGGAfCGAfUGfCGGGfCfCfUfUfCGfUfUfUGfUfUfUfCGfUfCfCAfCAGAfCGAfCfUfCGfCfCfCGAooooofCmAmAmAmAfUmGfUmA mAmAmAmAmAfCfU.

Bold: sticky sequence. fU and fC: 2′F modified pyrimidines. mA and mG:2′O methylated purines, o: C3 carbon linker.

P11, P7 and P6 are HSP90 binding aptamers capable of internalising uponbinding HSP90 at the cell surface.

The CCR5 binding aptamer G3 (SEQ ID NO:9) is described in Zhou et al.,2015, Chemistry & Biology 22, 379-390 Mar. 19, 2015 and in co-pendingU.S. patent application Ser. No. 14/801,710.

Formation of bi-specific aptamers capable of binding HSP90 and CCR5:P11, P7 or P6 conjugated to the sticky sequence via a polycarbon linkeris folded in binding buffer (phosphate-buffered saline solution [DPBS]without Ca²⁺ and Mg²⁺, 5 mM MgCl₂) at 95° C. for 5 mins and slowlycooled down. CCR5 aptamer conjugated to the sticky sequence via apolycarbon linker is folded in binding buffer (DPBS with MgCl₂ andCaCl₂) at 65° C. for 5 mins and slowly cooled down. The sameconcentration of P11, P7 or P6 and CCR5 aptamers is mixed and incubatedat 37° C. for 20 mins to make the bi-specific aptamer using ‘stickysequence’ technology.

Example 6: Bi-Specific Aptamers in Cancer Immunotherapeutics

To construct mortalin bi-specific RNA aptamers, truncated mortalinaptamer (tP19) was conjugated to CD3ε aptamer via complementary stickysequences (FIG. 22).

To construct mortalin-CD3ε bispecific aptamers, RNA aptamer against CD3εrecombinant protein was used for protein-SELEX. After 5 rounds of SELEX,deep-sequencing was performed to identify the CD3ε aptamers. Two CD3εaptamers were identified and named C3e2 and C3e3. The expected structurewas depicted by NUPACK (FIGS. 23A-23B). The identified sequences follow.

C3e2: (SEQ ID NO: 37) GGAGACAAGAAUAAACGCUCAAAUAGAAGCAGCAUCUUCCAAAUCAGUUUGUGUGUCCUCUAUUCGACAGGAGGCUCACAACAGGC C3e3: (SEQ ID NO: 38)GGGAGACAAGAAUAAACGCUCAAAUGCCUGUAGUUCGUAGCGAUUUAACUGCGUCAGUGAGGCUUCGACAGGAGGCUCACAACAGGC

For the functional assay, the binding of CD3ε aptamers to their targetswas determined by flow cytometry and confocal microscopy. The bindingassay of CD3ε aptamers to human and mice CD3ε was determined on humanCD3ε and mice CD3ε expressed HEK293 cells. C3e2 and C3e3 showed thecross-activity on both human CD3ε and mice CD3ε (FIGS. 24A-24B and25A-25B).

To determine the binding of CD3ε aptamers to their target again, Cy3labeled CD3ε aptamers were incubated on freshly isolated human T cellsand analyzed by flow cytometry. Both aptamers bound to human T cells,showing a shifted histogram (FIGS. 26A-26B). The sequences to constructmortalin bi-specific aptamers follow.

tP19: (SEQ ID NO: 33) 5′-GUGUAAUGUAGUAGUCoooooCUCAAUGGCGAAUGCCCGCCUAAUAGGG-3′ CD3e2; (SEQ ID NO: 34)5′GACUACUACAUUACACoooooGGGAGACAAGAAUAAACGCUCAAAUAGAAGCAGCAUCUUCCAAAUCAGUUUGUGUGUCCUCUAUUCGACAGGAGGCU CACAACAGGC-3′Bold: sticky sequence; o: C3 carbon linker CD3e3: (SEQ ID NO: 35)5′GACUACUACAUUACACoooooGGGAGACAAGAAUAAACGCUCAAAUGCCUGUAGUUCGUAGCGAUUUAACUGCGUCAGUGAGGCUUCGACAGGAGGCU CACAACAGGC-3′.Bold: sticky sequence; o: C3 carbon linker

Materials and Methods

CD3ε Aptamer SELEX

To isolated CD3ε aptamer, target proteins were purchased from Creativebiomart. The 2F′-RNA aptamers were selected from 40 nucleotide (nt)randomized sequences constructed by in vitro transcription of syntheticDNA templates with NTPs (2′F UTP, 2′F CTP, GTP, ATP, EpicentreBiotechnologies, Madison, Wis.) and T7 RNA polymerase. To remove RNAsthat bind nonspecifically to agarose beads, 1.44 μM of the RNA librarywas preincubated with 20 μl of Ni-NTA agarose beads in 100 μl bindingbuffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 2 mMdithiothreitol, and 1% BSA, 100 μg/mL yeast tRNA) for 30 min at roomtemperature with shaking, precipitated by centrifugation, and discarded.The precleared supernatant was transferred to a new tube and incubatedwith 300 nM of his-tagged human CD3ε (hCD3ε) for 30 min at roomtemperature. RNAs which bound to hCD3ε were recovered, amplified byRT-PCR and in vitro transcription, and used in the following selectionrounds. In subsequent rounds, hCD3ε concentration was reduced by 2-foldat every 2 round for more stringent condition. After 5 rounds of SELEX,the resulting cDNA was amplified. After 5 round of SELEX, deepsequencing was performed to identify the sequences. Two clone of CD3εaptamer were selected below. Structures of aptamers were predicted usingNUPACK using a salt correction algorithm and temperature correction for25° C.

Internalization Assay of CD3ε Aptamer

Stable cell lines expressing human or mouse CD3 were generated asfollows. Lentiviral vectors were constructed encoding fusions proteinsconsisting of EGFP and either full-length human CD3ε or full-lengthmouse CD3ε. Lentiviruses were amplified in HEK293T cells, harvested, andused to transfect and generate stable HEK293 cells. For binding studies,1×10⁵ cells of human or mouse CD3ε expressing HEK293 cells were seededin 35-mm glass-bottom dishes (MatTek, Ashland, Mass.) and grown inappropriate media for 24 hours. Aptamer RNAs were labeled with Cy3fluorescent dye using the Cy3 Silencer siRNA labeling kit (Thermo FisherScientific, Waltham, Mass.). Cy3-labeled aptamers folded in bindingbuffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 100 μg/mL yeasttRNA) were added to the cells at 200 nM and incubated for 2 hours.Before imaging, cells were washed with DPBS twice. Live-cell confocalimaging was performed with a Zeiss LSM 510 Meta inverted two-photonconfocal microscope system using a C-Apo 40×/1.2NA water immersionobjective, and AIM 4.2 software (Carl Zeiss, Jena, Germany). Hoechst33342 was used for counter-staining the cell nuclei.

Binding Assay of CD3ε Aptamer by Flow Cytometry

Human T cells (1×10⁵ cells/ml), freshly isolated using EasySep Human Tcell isolation kit (STEM CELL technologies), were incubated with 500 nMof Cy3-labeled CD3ε aptamer folded in binding buffer for 30 mins on ice.After washing with DPBS, DAPI (1 μg/mL) was added to exclude the deadcells and cells were immediately analyzed by flow cytometry.

What is claimed is:
 1. A bi-specific aptamer capable of binding a tumorcell antigen and an immune cell surface protein, wherein the bi-specificaptamer comprises a first aptamer complexed with a second aptamer, andfurther wherein the immune cell surface protein is CD8.
 2. Thebi-specific aptamer of claim 1, wherein the tumor cell antigen is TfR.3. The bi-specific aptamer of claim 1, wherein the bi-specific aptamercomprises a nucleic acid sequence having at least 80% sequence identityto SEQ ID NO:
 30. 4. The bi-specific aptamer of claim 1, wherein thebi-specific aptamer comprises a nucleic acid sequence having at least80% sequence identity to SEQ ID NO:
 28. 5. The bi-specific aptamer ofclaim 1, wherein one or more bases or nucleotides are chemicallymodified.
 6. The bi-specific aptamer of claim 1, wherein one or morenucleotides are chemically modified at the 2′ position of ribose.
 7. Acomplex of a bi-specific aptamer according to claim 1 and a tumor cellexpressing a tumor cell antigen to which the bi-specific aptamer iscapable of binding.
 8. A complex of a bi-specific aptamer according toclaim 1 and an immune cell expressing an immune cell surface protein towhich the bi-specific aptamer is capable of binding, wherein the immunecell surface protein is CD8.
 9. A complex of a bi-specific aptameraccording to claim 1, a tumor cell expressing a tumor cell antigen towhich the bi-specific aptamer is capable of binding, and an immune cellexpressing an immune cell surface protein to which the bi-specificaptamer is capable of binding, wherein the immune cell surface proteinis CD8.
 10. A complex of a bi-specific aptamer and an immune cellexpressing an immune cell surface protein to which the bi-specificaptamer is capable of binding, wherein: (a) the immune cell is a T-cell;(b) the bi-specific aptamer is capable of binding a tumor cell antigenand the immune cell surface protein; (c) the bi-specific aptamercomprises a first aptamer complexed with a second aptamer; and (d) thetumor cell antigen is TfR or the immune cell surface protein is CD8. 11.A pharmaceutical composition comprising a bi-specific aptamer accordingto claim 1 and a pharmaceutically acceptable carrier, diluent orexcipient.
 12. A bi-specific aptamer according to claim 1 for use in amethod of medical treatment.
 13. A bi-specific aptamer according toclaim 1 for use in a method of treatment of cancer.
 14. A bi-specificaptamer for use in a method of treatment of cancer, wherein: (a) thecancer is a pancreatic cancer; (b) the bi-specific aptamer is capable ofbinding a tumor cell antigen and the immune cell surface protein; (c)the bi-specific aptamer comprises a first aptamer complexed with asecond aptamer; and (d) the tumor cell antigen is TfR or the immune cellsurface protein is CD8.
 15. The bi-specific aptamer for use in a methodof treatment of cancer according to claim 13, wherein the canceroverexpresses at least one of HSP70, vimentin, HSP90, Tfr or PDGFR-a.16. The bi-specific aptamer of claim 1, wherein the bi-specific aptamercomprises a nucleic acid sequence having at least 80% sequence identityto SEQ ID NO:
 29. 17. The bi-specific aptamer of claim 1, wherein thetumor cell antigen is HSP70.
 18. The bi-specific aptamer of claim 1,wherein the tumor cell antigen is vimentin.
 19. The bi-specific aptamerof claim 1, wherein the tumor cell antigen is HSP90.
 20. The bi-specificaptamer of claim 1, wherein the tumor cell antigen is PDGFR.
 21. Thebi-specific aptamer of claim 1, wherein the bi-specific aptamercomprises the nucleic acid sequence of SEQ ID NO: 8.